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\begin{document}

\title{The Adenosine A\textsubscript{1} and A\textsubscript{2A} Receptor
C-termini are Necessary for Activation but not the Specificity of
Downstream Signaling}



\author[1]{Abhinav Jain}%
\author[1]{Claire McGraw}%
\author[1]{Anne Robinson}%
\affil[1]{Tulane University}%


\vspace{-1em}



  
  \date{June 12, 2024}


\begingroup
\let\center\flushleft
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\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Recent efforts to determine the high-resolution crystal structures for
the adenosine receptors (A1R and A2AR) have utilized modifications to
the native receptors in order to facilitate receptor crystallization and
structure determination. One common modification is a truncation of the
unstructured C-terminus, which has been utilized for all the adenosine
crystal structures obtained to date. However, the C-terminus has been
identified as a location for protein-protein interactions that may be
critical for physiological function of these important drug targets.
Here, we determine whether the presence of the full-length C-terminus
affected downstream signaling using a yeast MAPK response-based
fluorescence assay. Upon ligand binding, the A1\selectlanguage{greek}Δ\selectlanguage{english}291R or A2A\selectlanguage{greek}Δ\selectlanguage{english}316R
variants were unable to couple to human-yeast chimeric G-protein
chimeras to generate a downstream signal in yeast, though full-length
receptors showed native-like G-protein coupling. Further, constructs
transfected into mammalian cells (HEK-293) showed similar behavior --
i.e. the variants with C-terminal truncations lacked cAMP-linked
signaling compared to the full-length receptors. Although the C-terminus
was essential for G\selectlanguage{greek}α \selectlanguage{english}protein- associated signaling, chimeras of A1R with
a C-terminus of A2AR coupled to the A1R-specific G\selectlanguage{greek}α \selectlanguage{english}(i.e. G\selectlanguage{greek}α\selectlanguage{english}i1 versus
G\selectlanguage{greek}α\selectlanguage{english}s). This surprising result suggests that the C-terminus is important
in signaling, but not specificity, for the interaction with G\selectlanguage{greek}α \selectlanguage{english}protein.
This result has further implications in drug discovery both in enabling
the experimental use of chimeras for ligand design, and in cautious
interpretation of structure-based drug design based on truncated
receptors.%
\end{abstract}%



\sloppy


\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Receptor & Expression host & Ligand & Modification & Modification &
Modification & Modification & Reference\tabularnewline
\midrule
\endhead
& & & \textbf{Chimera} & \textbf{Stabilization} &
\textbf{Thermo-stabilization} & \textbf{Truncation} &\tabularnewline
A\textsubscript{2A}R & \emph{S. frugiperda} & ZM241385 & \textbf{X} & &
& \textbf{X} & Jaakola et al. (2008)\tabularnewline
& \emph{S. frugiperda} & \emph{UK-432097} & \textbf{X} & & & \textbf{X}
& Xu et al. (2011)\tabularnewline
& \emph{Trichoplusia ni} & \emph{Adenosine}; Synthetic ligand & & &
\textbf{X} & \textbf{X} & Lebon et al. (2011)\tabularnewline
& \emph{S. frugiperda} & Caffeine; ZM241385; XAC & & & \textbf{X} &
\textbf{X} & Dore et al. (2011)\tabularnewline
& \emph{P. pastoris} & ZM241385 & & \textbf{X} & & \textbf{X} & Hino et
al. (2012)\tabularnewline
& \emph{S. frugiperda} & Novel compound & & & \textbf{X} & \textbf{X} &
Congreve et al. (2012)\tabularnewline
& \emph{S. frugiperda} & ZM241385 & \textbf{X} & & & \textbf{X} & Liu et
al. (2012)\tabularnewline
& \emph{Trichoplusia ni} & \emph{CGS21680} & & & \textbf{X} & \textbf{X}
& Lebon et al. (2015)\tabularnewline
& \emph{Trichoplusia ni} & ZM241385 and novel compounds & \textbf{X} & &
\textbf{X} & \textbf{X} & Segala et al. (2016)\tabularnewline
& \emph{Trichoplusia ni} & \emph{NECA} & \textbf{X} & \textbf{X} & &
\textbf{X} & Carpenter et al. (2016)\tabularnewline
& \emph{S. frugiperda} & ZM241385 & \textbf{X} & & & \textbf{X} & Batyuk
et al. (2016)\tabularnewline
& \emph{S. frugiperda} & Novel compound & \textbf{X} & & & \textbf{X} &
Sun et al. (2017)\tabularnewline
& \emph{S. frugiperda} & ZM241385 & \textbf{X} & & & \textbf{X} &
Martin-Garcia et al. (2017)\tabularnewline
& \emph{S. frugiperda} & ZM241385 & \textbf{X} & & & \textbf{X} &
Melnikov et al. (2017)\tabularnewline
& \emph{Trichoplusia ni} & Theophylline; caffeine; PSB36 & \textbf{X} &
& \textbf{X} & \textbf{X} & Cheng et al. (2017)\tabularnewline
& \emph{Trichoplusia ni} & ZM241385 & \textbf{X} & & \textbf{X} &
\textbf{X} & Weinert et al. (2017)\tabularnewline
& \emph{S. frugiperda} & ZM241385 & \textbf{X} & & \textbf{X} &
\textbf{X} & Broecker et al. (2018)\tabularnewline
& \emph{P. pastoris} & ZM241385 & \textbf{X} & & & \textbf{X} & Eddy et
al. (2018)\tabularnewline
& \emph{Trichoplusia ni} & Theophylline and novel compounds & \textbf{X}
& & \textbf{X} & \textbf{X} & Rucktooa et al. (2018)\tabularnewline
& \emph{P. pastoris} & \emph{UK-432097} & \textbf{X} & & & \textbf{X} &
White et al. (2018)\tabularnewline
& \emph{Trichoplusia ni} & \emph{NECA} & \textbf{X} & \textbf{X} & &
\textbf{X} & Garcia-Nafria et al. (2018)\tabularnewline
& \emph{S. frugiperda} & ZM241385 & \textbf{X} & & & \textbf{X} &
(Martin-Garcia et al., 2019)\tabularnewline
A\textsubscript{1}R & \emph{S. frugiperda} & DU172 & \textbf{X} & & &
\textbf{X} & Glukhova et al. (2017)\tabularnewline
& \emph{Trichoplusia ni} & PSB36 & \textbf{X} & & \textbf{X} &
\textbf{X} & Cheng et al. (2017)\tabularnewline
& \emph{Trichoplusia ni} & \emph{Adenosine} and DU172 & \textbf{X} & & &
& Draper-Joyce et al. (2018)\tabularnewline
\bottomrule
\end{longtable}

Receptor chimeras have traditionally been used to understand the role of
the receptor domains in ligand recognition, G-protein coupling and
specificity, and the ability to produce downstream signaling. In our
previous study (Jain et al., 2018), we created an adenosine
A\textsubscript{1}/A\textsubscript{2A} receptor chimera to improve
membrane localization and expression in yeast for
A\textsubscript{1}receptor (A\textsubscript{1}R) variants and reported
exceptional yields of the active receptor compared to parental
A\textsubscript{1}R expressed in any host system to date.

Adenosine receptors are a GPCR subfamily of four receptors
(A\textsubscript{1}R, A\textsubscript{2A}R, A\textsubscript{2B}R and
A\textsubscript{3}R) that recognize the natural ligand adenosine, an
important energy metabolite (Fredholm et al., 2001; Fredholm et al.,
2011). Adenosine is produced in tissues under stressful conditions like
ischemia or hypoxia or energy ``demand-supply'' imbalance (Fredholm et
al., 2005; McIntosh et al., 2012). All four adenosine receptor subtypes
provide critical protection under stressful conditions and therefore,
are therapeutic targets for Parkinson's disease, Alzheimer's disease,
cardiovascular diseases, and many others (Chen et al., 2013). Multiple
crystal structures of A\textsubscript{2A}R have been resolved with bound
agonists or antagonists (Table 1). Recently, three crystal structures
have been reported for A\textsubscript{1}R (Cheng et al., 2017;
Draper-Joyce et al., 2018; Glukhova et al., 2017). All structures
reported for the adenosine receptors contain a C-terminal truncation,
except a recently published cryo-EM structure of A\textsubscript{1}R
(Draper-Joyce et al., 2018). The C-terminus of A\textsubscript{1}R is 34
amino acids long, whereas the A\textsubscript{2A}R C-terminus is
relatively long with 122 amino acids. The two crystal structures of
A\textsubscript{1}R contain a truncation from residues 311 and 316. Most
crystal structures of A\textsubscript{2A}R contain a truncation from
residue 316 (A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R), corresponding to only 26 out of
the 120 aminos acids or approximately 20\% of the total
A\textsubscript{2A}R C-terminus. The long C-terminus of
A\textsubscript{2A}R has been hypothesized to be involved in receptor
expression (Britton, 2012; Jain et al., 2018; Moriyama et al., 2010),
interactions with other signaling partners (Gsandtner et al., 2006;
Zezula et al., 2008), oligomerization (Navarro et al., 2018) and
receptor turnover (Singh et al., 2010; Weiss et al., 2002). However,
previous studies have suggested that the A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R has
native-like signaling (Bennett et al., 2013; Klinger et al., 2002;
Palmer et al., 1997).

Yeast share many functionally exchangeable proteins involved in the GPCR
signaling pathway with higher eukaryotes (Dohlman et al., 1991; Elion,
2000), and have served as a useful microbial platform for rapid ligand
screening and lead development for orphan GPCRs (Huang et al., 2015).
The GPCR-mediated pathway in yeast is responsive to the presence of
peptide mating pheromones that regulate metabolism related to mating.
Activated receptors catalyze dissociation of Gpa1, the yeast G protein,
activating a mitogen-activated protein kinase (MAPK) cascade, which has
been used as a unique platform to study human GPCR signaling (King et
al., 1990). In contrast, the presence of multiple GPCRs and
G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}proteins in native mammalian systems can confound the
results from downstream signaling assays =. Yeast provides a relatively
simple and inexpensive platform without the complexities of multiple
GPCRs, receptor promiscuity, and crosstalk that occurs in native
mammalian hosts (Chen et al., 2007; Saito, 2010).

Engineered yeast strains with modification to the native MAPK-based
signaling pathway to report on ligand-mediated downstream signaling from
human GPCRs (Figure 1A) were obtained both from the Broach laboratory
(Fowlkes et al., 1997) and the Dowell laboratory at GlaxoSmithKline
(GSK, Brentford, UK) (Brown et al., 2000). In these yeast strains, the
last five amino acids of native yeast G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}(Gpa1) were
replaced with the last five amino acids residues from a human
G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}to yield native-like
GPCR-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}interactions. This replacement has been shown to
be sufficient for coupling with many human GPCRs, including human
A\textsubscript{2A}R, resulting in a native-like dose reponse and ligand
binding order preference (Brown et al., 2000). Because of
structure-based drug discovery efforts that rely on truncated receptors
for in silico screening, we investgated one of the key protein-protein
interactions of the C-terminus, coupling to G-protein to activate
downstream signaling, by utilizing this engineered yeast pheromone
response pathway. In addition, the results were validated in transiently
transfected mammalian cells to provide further evidence of the value of
screening these signaling pathways in yeast.

\textbf{Materials and Methods}

\emph{2.1 Materials}

Adenosine receptor ligands NECA (5'-N-ethylcarboxamidoadenosine), CPA
(N\textsuperscript{6}-cyclopentyladenosine) and CGS21680 were purchased
from Tocris (Minneapolis, MN). Forskolin was obtained from Sigma-Aldrich
(St. Louis, MO). Precision Plus Protein Western C Standards was
purchased from Biorad (Hercules, CA). Human embryonic kidney cells
(HEK-293; ATCC), Dulbecco's modified eagle medium (DMEM, 11995-065),
Opti-MEM I reduced serum media (31985-070), fetal bovine serum (FBS,
16000-044), Lipofectamine 2000 transfection reagent (11668-019), RIPA
buffer, Halt Protease and Phosphatase Inhibitor Cocktail, mammalian
expression vectors (pCEP4) and Alexa 568- donkey anti-rabbit antibody
(A10042) were obtained from Invitrogen Life Technologies (Carlsbad, CA).
The cAMP dynamic 2 kit was purchased from Cisbio US Inc (Bedford, MA).
The mouse monoclonal A\textsubscript{2A}R antibody was obtained from
Santa Cruz Biotechnology (sc-32261, Dallas, TX). The rabbit anti-GFP
antibody (ab6556) and goat pAb to Mouse IgG HRP antibody (ab97265) were
obtained from Abcam (Cambridge, MA).

\emph{2.2 Strains and culture conditions}

\emph{E. coli} strain DH5\selectlanguage{greek}α \selectlanguage{english}was used for amplifying yeast expression
plasmids and mammalian expression vectors. \emph{E. coli} was grown in
Luria-Bertani media supplemented with 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL ampicillin at 37\selectlanguage{ngerman}°C at
250 rpm.

\textbf{Table 2.} List of yeast strains used.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Yeast Strain} & \textbf{G protein} & \textbf{Last 5 amino acids
at C-terminal} & \selectlanguage{greek}\textbf{Equivalent human
G\textsubscript{α}}\selectlanguage{english}\tabularnewline
\midrule
\endhead
MMY12, BY4741 & Gpa1 & KIGII\textsuperscript{COOH} & GPA1
(yeast)\tabularnewline
MMY14 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}q(5) & EYNLV\textsuperscript{COOH} &
GNAQ, GNA11\tabularnewline
MMY16 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}16(5) & EINLL\textsuperscript{COOH} &
GNA15, GNA16\tabularnewline
MMY19 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}12(5) & DIMLQ\textsuperscript{COOH} &
GNA12\tabularnewline
MMY20 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}13(5) & QLMLQ\textsuperscript{COOH} &
GNA13\tabularnewline
MMY21 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}14(5) & EFNLV\textsuperscript{COOH} &
GNA14\tabularnewline
MMY22 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}o(5) & GCGLY\textsuperscript{COOH} &
GNAO\tabularnewline
MMY23, CY13393 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i1(5) &
DCGLF\textsuperscript{COOH} & GNAI1, GNAI2, GNAT1, GNAT2,
GNAT3\tabularnewline
MMY24 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i3(5) & ECGLY\textsuperscript{COOH} &
GNAI3\tabularnewline
MMY25 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}z(5) & YIGLC\textsuperscript{COOH} &
GNAZ\tabularnewline
MMY28, CY13399 & Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s(5) &
QYELL\textsuperscript{COOH} & GNAS, GNAL\tabularnewline
\bottomrule
\end{longtable}

All yeast strains used in this study are summarized in Table 2. Yeast
strains with modified pheromone response pathway and human-yeast
chimeric G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}proteins (Fig 1A) were obtained from the
Broach laboratory (Fowlkes et al., 1997) and Glaxo-Smith-Kline (GSK)
(Brown et al., 2000). These parental yeast strains were grown in YPD
media (2\% bacto peptone, 2\% glucose, 1\% yeast extract) and depending
on the fus1 transformation, supplemented with 300 \selectlanguage{greek}µ\selectlanguage{english}g/mL hygromycin B or
200 \selectlanguage{greek}µ\selectlanguage{english}g/mL G418. Yeast expression plasmids were constructed using
homologous recombination in \emph{S. cerevisiae} strain BY4741
(\selectlanguage{greek}\emph{MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0}\selectlanguage{english} ) and were grown in synthetic
media. The synthetic media (SD or SG) was comprised of 2\% dextrose or
galactose, respectively, 0.67\% yeast nitrogen base, citrate buffer at
pH 5.4 (4.2 g/L citric acid and 14.7 g/L sodium citrate) and
supplemented with amino acids and essential nutrients per Burke et al.
(2000) (Burke et al., 2000). Uracil was omitted from this media (SD-ura
or SG-ura) to select for plasmid-containing cells. Yeast was grown in
culture tubes and multiwell plates at 30\selectlanguage{ngerman}°C at 275 rpm.

Human embryonic kidney (HEK-293) cells were maintained in growth media
containing DMEM with 10\% FBS at 37 °C in a 5\%
CO\textsubscript{2}incubator. Transient transfections were performed by
seeding cells on day 0 to be approximately 70\% confluent on day 1. On
day 1, cells were transfected using 10 \selectlanguage{greek}μ\selectlanguage{english}L Lipofectamine 2000 reagent,
and 1 \selectlanguage{greek}μ\selectlanguage{english}g DNA in two mL Opti-MEM reduced serum media (per 25
cm\textsuperscript{2} flask). On day 2 cells were placed back in growth
media, and used for experimentation on day 3, approximately 36 hours
post-transfection. The cAMP accumulation assay described below (Section
2.6) was performed on cells with passage number less than 25.

\emph{2.3 Yeast genomic transformation}

To develop a fluorescence-based assay to measure the downstream
signaling response in yeast following ligand binding, monomeric Cherry
fluorescent protein (mCherry) (Shaner et al., 2004) was introduced into
the FUS1 locus under control of the FUS1 promoter. To this end,
overlapping fragments were first assembled in yeast using homologous
recombination as described below. The fragment consisted of the mCherry
fluorescent protein and hygromycin resistance gene hphMx6 or kanamycin
resistance gene KanR2 with the translation elongation factor 1 promoter
and terminator (pTEF and TEFt). The fragment was flanked with
approximately 300 base pairs of the Fus1 promoter and Fus1 terminator to
aid in genomic recombination. The Fus1 promoter and Fus1 terminator
sequences were amplified from BY4741 using colony PCR. The mCherry
protein and pTEF-hphMx6-TEFt fragments were amplified from the pBS35
plasmid, while the pTEF-KanR2-TEFt fragment was amplified from the pBS7
plasmid (Figure 1B). Both pBS7 and pBS35 were received from the Yeast
Resource Center at the University of Washington. The fragments were
assembled in BY4741 using homologous recombination using pRS316 as a
template. Fragment assembly was verified using Sanger sequencing
(Operon, Louisville, KY). The resulting fragment was then amplified
using PCR and transformed into yeast using the protocol from Gietz and
Woods (Gietz et al., 2002). Colony PCR was used to confirm successful
genomic integration. Partial sequencing confirmation of final clones was
obtained for some of the transformants.

\emph{2.4 Subcloning and plasmid construction}

A set of yeast expression plasmids (Table 3) containing a GPCR and
C-terminal protein tags, necessary for adenosine receptor detection and
quantification, was constructed using homologous recombination in BY4741
as described previously (Jain et al., 2018). The plasmid contains a
galactose (pGAL\textsubscript{1-10}) promoter, a pre-pro leader sequence
(PP) (Arnold et al., 1998) for targeting to the secretory pathway and
the CYC1 terminator (CYC1\textsubscript{t}) (Jain et al., 2018). For
fluorescence microscopy, the GPCRs were C-terminally tagged for easier
detection of protein expression with monomeric Citrine fluorescent
protein (mCitrine) (Young et al., 2012). Single-point
A\textsubscript{2A}R mutants were created using the QuikChange II
Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA).
Sequencing was used to confirm the correct gene sequence for the
constructs (Operon, Louisville, KY).

\textbf{Table 3.} List of plasmids used for receptor expression in yeast
and mammalian cells. Yeast expression plasmids contain an N-terminal
leader sequence (PP) to improve receptor expression and trafficking to
the plasma membrane (Arnold et al., 1998).\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Name} & \textbf{Plasmid}\tabularnewline
\midrule
\endhead
ARJ001 & pRS316 pGal\textsubscript{1-10} PP A\textsubscript{1}R mCit
cyc\textsubscript{t}\tabularnewline
ARJ089 & pRS316 pGal\textsubscript{1-10} PP A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R mCit
cyc\textsubscript{t}\tabularnewline
ARJ002 & pRS316 pGal\textsubscript{1-10} PP
A\textsubscript{1}/A\textsubscript{2A}R mCit
cyc\textsubscript{t}\tabularnewline
ARJ051 & pRS316 pGal\textsubscript{1-10} PP
A\textsubscript{1}/A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R mCit
cyc\textsubscript{t}\tabularnewline
ARJ030 & pRS316 pGal\textsubscript{1-10} PP A\textsubscript{2A}R mCit
cyc\textsubscript{t}\tabularnewline
ARJ057 & pRS316 pGal\textsubscript{1-10} PP A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R
mCit cyc\textsubscript{t}\tabularnewline
ARJ194 & pCEP4 A\textsubscript{1}R\tabularnewline
ARJ195 & pCEP4 A\textsubscript{1}/A\textsubscript{2A}R\tabularnewline
ARJ196 & pCEP4
A\textsubscript{1}/A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R\tabularnewline
ARJ320 & pCEP4 A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R\tabularnewline
CM001 & pCEP4 A\textsubscript{2A}R\tabularnewline
CM002 & pCEP4 A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R\tabularnewline
ARJ073 & pRS316 pGal\textsubscript{1-10} PP pFus1 mcherry
pTEF-kanR2-tTEF Fus1\textsubscript{t}
cyc\textsubscript{t}\tabularnewline
ARJ172 & pRS316 pGal\textsubscript{1-10} PP pFus1 mcherry
pTEF-hphMx6-tTEF Fus1\textsubscript{t}
cyc\textsubscript{t}\tabularnewline
\bottomrule
\end{longtable}

Mammalian expression vector pCEP4 was used for expressing receptors in
transiently transfected HEK-293 cells. Untagged A\textsubscript{1}R,
A\textsubscript{1}/A\textsubscript{2A}R, and A\textsubscript{1}[?]291R
were inserted into the pCEP4 multiple cloning site between HindIII and
NotI restriction enzyme sites, whereas
A\textsubscript{1}/A\textsubscript{2A}[?]316R, A\textsubscript{2A}R, and
A\textsubscript{2A}[?]316R were inserted between KpnI and XhoI restriction
enzyme sites. Transformations of \emph{E. coli} were performed by the
heat shock method. Sequencing was used to confirm the correct gene
sequences for the plasmids (Operon, Louisville, KY).

\emph{2.5 MAPK response signal determination}

All ligand stock solutions were prepared to the highest soluble
concentration (typically around 40-100mM) in dimethyl sulfoxide (DMSO),
according to the recommendations of the manufacturer. Working
concentrations of 5 mM ligand (50x) in DMSO were used for all yeast
signaling experiments. Yeast cultures were grown overnight in SD-ura
selection in 400\selectlanguage{greek}µ\selectlanguage{english}L or 1mL media in 48-well or 24-well plates (Falcon
353047 and 353078, Corning, NY), respectively, at 30\selectlanguage{ngerman}°C at 275 rpm.
Recombinant GPCR expression was induced by transferring 12.5 \selectlanguage{greek}µ\selectlanguage{english}L of
overnight culture into 400 \selectlanguage{greek}µ\selectlanguage{english}L SG-ura. For some strains 0.125\% glucose
was used to supplement the SG-ura media to improve cell growth of the
engineered yeast strains. This level of glucose supplementation has been
shown to result in minimal glucose-based suppression of the galactose
promoter, as described previously (Bitter et al., 1988). After 24 hours
of GPCR expression, twelve \selectlanguage{greek}µ\selectlanguage{english}L of the overnight culture was added to 380
\selectlanguage{greek}µ\selectlanguage{english}L fresh SG-ura media per well of a 48-well plate. Eight \selectlanguage{greek}µ\selectlanguage{english}L of ligand or
DMSO was added to each well (final DMSO concentration at 2\% (v/v) per
well). A high ligand concentration has been shown previously to be
needed for effective downstream signaling in yeast (Hara et al., 2012;
Niebauer et al., 2005; Price et al., 1995; Price et al., 1996). After
ligand addition, the 48-well plate was incubated at 30 \selectlanguage{ngerman}°C at 275 rpm for
24 hours. Adenosine deaminase treatment was not required for working
with the yeast-based assay as previous studies show this treatment does
not impact downstream signaling measurements (Bertheleme et al., 2013;
Peeters et al., 2012). Similar results were obtained from 4-hour
incubations, but the signal:noise ratio was not as pronounced.
Fluorescence intensities of 100 \selectlanguage{greek}µ\selectlanguage{english}L of resulting liquid culture were
measured in triplicate in a 96-well plate (Costar 3915, Corning, NY)
using a BioTek Synergy H1 microplate reader (Winooski, VT) maintained at
30 \selectlanguage{ngerman}°C. Experiments were performed for six independent transformants.

\emph{2.6 Cyclic adenosine monophosphate accumulation assay}

The cyclic adenosine monophosphate (cAMP) accumulation assay was
performed as previously described in McGraw et al. (2019) (McGraw et
al., 2019). Briefly, transiently transfected HEK-293 and control cells
were incubated for 30 minutes in the presence or absence of ligand at a
cell density of 1,000 cells/well in a white 384 well plate (Grenier
Bio-One \#784075, Monroe, NC). Excess cells were pelleted and stored at
-80 °C for subsequent Western blotting. The concentration of cAMP per
well was determined using the cAMP dynamic 2 kit using a BioTek Synergy
H1 Plate Reader according to the manufacturer's protocol. Our previous
study (McGraw et al., 2019) has shown that adenosine deaminase (ADA)
pre-treatment of cells did not alter the ligand binding or downstream
signaling, and therefore the cells were not treated with ADA prior to
ligand treatment while utilizing the CisBio HTRF kits (McGraw et al.,
2019). Experiments were performed in triplicates for three independent
transfections. Data was analyzed as per manufacturer's recommendation
and mean and standard error were plotted using Prism (GraphPad, La
Jolla, CA). Student's t-test was performed using Prism to obtain the
significance of the data.

\emph{2.7 Western blotting}

Yeast cell pellets (10 OD\textsubscript{600}) were resuspended in 250 \selectlanguage{greek}µ\selectlanguage{english}L
lysis buffer (10\% glycerol, 50 mM sodium phosphate, 300 mM sodium
chloride, pH 8) supplemented with cOmplete EDTA-free protease inhibitor
cocktail (Roche, Indianapolis, IN). An equal volume of 0.5 mm
zirconia/silica beads (BioSpec, Bartlesville, OK) was added to the cells
and lysis was performed using a vortexer or a BeadBug homogenizer
(Benchmark Scientific, Edison, NJ). Cell lysates were combined with 4X
Laemmli sample loading buffer supplemented with \selectlanguage{greek}β-\selectlanguage{english}mercaptoethanol
(Bio-Rad, Hercules, CA). One OD\textsubscript{600} equivalent of cell
lysate was loaded per well for Western blotting. Precision Plus Protein
WesternC Standard (BioRad)was used as a standard to enable molecular
weight estimation. Rabbit anti-GFP antibody (1:5000 dilution) and Alexa
568-donkey anti-rabbit (1:2500) was used to detect mCitrine
protein-tagged receptors.

Transiently transfected HEK-293 cells were scraped, pelleted, and
resuspended in ice-cold 1X TE buffer (1\% 1M Tris-Cl pH 7.5, 0.2\% 500mM
EDTA pH 8) with protease inhibitors. Cells were sonicated with a Branson
Sonifier 450 at 50\% power for 30 pulses and then centrifuged at 2,000
xg for 5 min at 4\selectlanguage{ngerman}°C to remove cell debris and unlysed cells. The
supernatant was then centrifuged at 100,000 x g for 1 hr at 4°C to
pellet cell membranes. Membranes were solubilized in 1X RIPA buffer (10
mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1\% Triton X-100, 0.1\%
sodium deoxycholate, 0.1\% SDS, 140 mM NaCl) with protease inhibitors;
if necessary, membranes were sonicated again for 5 pulses at 50\% power
to break up any visible pieces of membrane. BCA assay (Pierce; Rockford,
IL) was performed to determine the total protein concentration of
isolated membrane, using bovine serum albumin (BSA; Thermo Fisher,
Waltham, MA) as a standard.

Isolated HEK cell membranes were utilized for A\textsubscript{2A}R and
A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R protein quantification via Western immunoassay.
Western blotting analysis could not be performed for A\textsubscript{1}R
and its variant due to the lack of an effective antibody against the
receptor. 10 \selectlanguage{greek}μ\selectlanguage{english}g of total protein per sample was loaded onto a 12\%
Tris-Glycine gel and electrophoresed in SDS buffer at 125V for 65
minutes. Western immunoassay was performed using adenosine
A\textsubscript{2A}R mouse monoclonal IgG antibody (sc-32261, Santa Cruz
Biotechnology, Dallas, TX) at 1:5000 dilution, and Goat pAb to Mouse IgG
HRP antibody at 1:5000 dilution. Membranes were imaged with the UVP
BioSpectrum imaging system.

\textbf{3. Results}

\selectlanguage{greek}\emph{3.1 Whole cell fluorescence to monitor ligand-mediated GPCR-
G\textsubscript{α} interaction}\selectlanguage{english}

Because the yeast G protein-couled signaling pathway contains homologues
to proteins in the human signaling pathway, engineered yeast have been
used to successfully recapitulate native ligand binding preferences and
G protein coupling for human GPCRs (Bertheleme et al., 2013; Brown et
al., 2000; Fowlkes et al., 1997; Peeters et al., 2012). To further the
utility of these previously engineered yeast strains, we replaced the
original Fus1 modification that relied on growth-dependent signaling
(via His3 expression) in the Broach lab strain (Fowlkes et al., 1997) or
\selectlanguage{greek}β-\selectlanguage{english}galactosidase reporter activity in the GSK strains (Brown et al.,
2000) with an easily detectable fluorescence signal, monomeric Cherry
fluorescent protein (mCherry). mCherry is produced in the cells upon
ligand-mediated downstream signal activation via the human
GPCR-G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}protein coupling (Figure~1A). The signal/noise
ratio of mCherry fluorescence in these strains can be easily compared by
addition of agonist relative to a control. Here, each yeast strain
(Table 2) acts as an independent downstream signaling reporter for a
GPCR-G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}interaction.

Twelve strains containing different yeast-human
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}chimeras reproduce downstream signaling responses of
human G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}proteins (Brown et al., 2000)\textbf{.} These
strains can be classified into five G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}families:
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i/o, G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s, G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}q,
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}12 and native G\selectlanguage{greek}\textsubscript{α}. \selectlanguage{english}With the modified
G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}chimera strains, we successfully mapped the
interaction between the A\textsubscript{1} adenosine receptors and the
appropriate G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}using the non-selective high-affinity
adenosine receptor family agonist, NECA (100 \selectlanguage{greek}µ\selectlanguage{english}M, Figure 1C).
A\textsubscript{1}R showed a signaling response upon agonist binding
with the inhibitory G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}family (G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i1,
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i3, G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}o and G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}z) and
the promiscuous G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}16. The highest signal was observed
for the yeast strain expressing the Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}o chimera.
Note that ligand levels are well above expected
K\textsubscript{D}values; however, this behavior is consistent with
earlier studies (Bertheleme et al., 2013; Brown et al., 2000; Peeters et
al., 2012; Stewart et al., 2009), and perhaps reflects ligand incapable
of penetrating the chitosan-rich yeast cell wall to reach the plasma
membrane, resulting in an apparent reduced effective ligand
concentration at the membrane.

To ensure our results were not strain-dependent, we compared the
signaling response obtained from engineered yeast strains modified from
those of the Broach laboratory. A\textsubscript{1}R and
A\textsubscript{2A}R were expressed in yeast strains expressing
Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i1 and Gpa1-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s. Both receptors
maintained their native G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}coupling-specificity, as
observed in Figure 1C for A\textsubscript{1}R signaling in the GSK
strains. The Broach laboratory strains showed a higher signal/noise
ratio compared to the GSK strains for both receptors (Figure 2 A \& B).
Because of the higher signal/noise ratio compared to the GSK strains,
the Broach strains were utilized for subsequent studies investigating
the role of the C-terminus in downstream signaling.

\emph{3.2 Loss of the cytoplasmic C-terminus results in loss of
downstream signaling}

To investigate the role of the C-terminus on downstream signaling,
truncated A\textsubscript{1}R and A\textsubscript{2A}R were constructed.
A\textsubscript{1}R was truncated at residue
291(A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R), corresponding with the end of
transmembrane 7 and the start of the cytoplasmic tail; this
A\textsubscript{1}R truncation lacks helix 8. The A\textsubscript{2A}R
truncation at residue 316 (A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R) was constructed
based on the agonist-bound crystal structure of the receptor obtained by
Lebon et al. (Lebon et al., 2011). This A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R
contains the helix loop 8 and some residues of the cytoplasmic tail, and
has been reported previously to have native-like affinity for the
agonist NECA and antagonist ZM 241385 by Magnani and colleages (Magnani
et al., 2008). Expression of the truncated receptors with C-terminal
tagged mCitrine fluorescent protein fusions was confirmed using Western
blot analysis (Figure 3A). Previous studies from our laboratory show
that the C-terminal fluorescent protein fusion does not impact
trafficking or activity of A\textsubscript{2A}R (Niebauer et al., 2006;
Niebauer et al., 2004; O'Malley, 2009; Wedekind et al., 2006).To
evaluate the localization of these receptors inside the yeast, confocal
microscopy was performed (Figure 3B ii and iv). Both full-length and
truncated A\textsubscript{2A}R showed efficient localization of the
receptor to the plasma membrane, whereas both A\textsubscript{1}R
constructs showed puncta inside the cell with minimal receptor located
at the cell periphery (Figure 3B i and iii).

Upon agonist binding, there was no downstream signaling observed in the
truncated A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R and A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R as
compared to their full-length receptors in the inhibitory or stimulatory
yeast strains (Figure 3C and 3D). Similarly, a previous study performed
on rat A\textsubscript{1}R had identified that the loss of the
C-terminus resulted in a loss of downstream signaling (Pankevych et al.,
2003). But, the loss of signaling for the truncated
A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R was surprising, as the agonist-bound crystal
structures have been reported to be in an active state (Carpenter et
al., 2016; Lebon et al., 2011), and the truncation localizes well to the
cell surface (as shown in Fig 3B and reported by our lab previously in
(Jain et al., 2018)) and binds ligand in mammalian cells (Magnani et
al., 2008).

To validate that our observations in yeast reflect native-like behavior,
we assayed cyclic adenosine monophosphate (cAMP) accumulation following
ligand addition in mammalian cells, as described in the Materials and
Methods. HEK-293 cells were transiently transfected with pCEP4 encoding
full-length or truncated receptor using lipofectamine.
A\textsubscript{2A}R couples to G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s, and thus agonist
binding activates adenylyl cyclase, resulting in cAMP synthesis. As
expected, cells transfected with empty plasmid showed negligible cAMP
synthesis in the absence of ligand and remained unchanged following the
addition of a selective A\textsubscript{2A}R agonist, CGS21680 (1 \selectlanguage{greek}μ\selectlanguage{english}M)
(Figure 4A). The presence of the full-length A\textsubscript{2A}R led to
constitutive activation in the absence of ligand as well as a
significant increase in cAMP levels following agonist treatment,
consistent with previous studies (McGraw et al., 2019). As expected,
based on the yeast results, the A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R showed no
increase in cAMP levels upon agonist addition. These data show that
A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R does not activate G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s,
suggesting the C-terminus is necessary for downstream signaling of the
receptor.

Western blot analysis of membrane preparations was utilized to verify
that the absence of A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R activity was not due to
reduced protein expression. Similar levels of A\textsubscript{2A}R and
A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R expression were detected in HEK-293 cells;
therefore, the lack of a C-terminus had no effect on protein expression
levels in cell lysates or extracted total membrane fractions (Figure
4B). Non-transfected cells did not show any receptor expression via
western blot analysis (data not shown).

A\textsubscript{1}R couples to G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i/o, which inhibits
activation of adenylyl cyclase, so in the absence of ligand there should
be minimal changes to cAMP levels, consistent with our results (Fig. 5).
Forskolin directly activates adenylyl cyclase, which leads to
stimulation of the production of cAMP even in cells not expressing
A\textsubscript{1}R, so treatment with 10 \selectlanguage{greek}μ\selectlanguage{english}M forskolin was used to
elevate the basal level of cAMP. Cells expressing A\textsubscript{1}R
showed a reduction in cAMP following treatment with an
A\textsubscript{1}R-selective agonist (1 \selectlanguage{greek}μ\selectlanguage{english}M CPA in the presence of 10 \selectlanguage{greek}μ\selectlanguage{english}M
forskolin). In cells expressing A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R, activation of
adenylyl cyclase via forskolin treatment led to the synthesis of cAMP,
while agonist treatment showed a negligible change in cAMP levels, in
contrast to full-length A\textsubscript{1}R. The A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R
behaved similarly to the empty plasmid control, as well as
non-transfected cells (not shown). This data along with data obtained
from engineered yeast strains show that A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R does not
activate G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i/o following ligand treatment, suggesting
the C-terminus is necessary for downstream signaling of the
A\textsubscript{1}R.

The observations for both adenosine receptors are consistent with our
results from the yeast pheromone response. Our observation is important
as the yeast system can be utilized to screen and validate receptor
variant activity. Here, it suggests that the crystal structures of
A\textsubscript{2A}R with agonists that have all been resolved with the
\selectlanguage{greek}Δ\selectlanguage{english}316 truncation may not represent the fully active state of the
receptors; that is, the region following residue 316 is necessary for
interaction with the G protein.

\selectlanguage{greek}\emph{3.3 The C-terminus does not play a role in the specificity of
G\textsubscript{α} coupling}\selectlanguage{english}

To understand the role of the C-terminus in the specificity of the
GPCR-G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}interaction, we constructed an
A\textsubscript{1}/A\textsubscript{2A}R chimera with all seven
transmembrane domains of A\textsubscript{1}R (residues 1-290) and the
C-terminus of the A\textsubscript{2A}R (residues 291-412) using
homologous recombination. The crystal structures of A\textsubscript{2A}R
have been resolved with a truncation at the
316\textsuperscript{th}residue, and therefore, a truncated chimera
(A\textsubscript{1}/A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R) consisting of the
transmembrane domains of A\textsubscript{1}R and the C-terminus of
A\textsubscript{2A}R truncated at the 316\textsuperscript{th} residue
was constructed. This truncated chimera contains the helix 8 residues of
A\textsubscript{2A}R. This A\textsubscript{2A}R truncation at residue
316 has been previously reported to have native-like binding to the
agonist NECA and the antagonist ZM 241385 by Magnani and colleages
(2008) at 32 nM and 12 nM, respectively, compared to 20 nM and 1 nM for
the wild-type receptor reported previously (de Lera Ruiz et al., 2014).
Full-length expression of the chimeras was observed using Western blot
analysis (Figure 6A) and showed slightly improved membrane localization
to the plasma membrane (Figure 6Bii) as compared to wild-type
A\textsubscript{1} receptor (Figure~3Bi).

The downstream signaling response was evaluated in the inhibitory and
stimulatory yeast reporter strains, and both the full-length and
truncated chimeras, respectively, showed coupling with the inhibitory
yeast strain, similar to wild-type A\textsubscript{1}R (Figure 6C). The
truncated chimera showed reduced MAPK signaling via lower mCherry levels
than the full-length chimera. No signaling response was obtained in the
stimulatory yeast strains for the A\textsubscript{1}R variants (Figure
6D). This observation suggests that the presence of the
A\textsubscript{2A}R C-terminus does not affect the interaction of the
chimeric A\textsubscript{1}/A\textsubscript{2A} receptor with the
native-like inhibitory G\selectlanguage{greek}\textsubscript{α}. \selectlanguage{english}This observation is
consistent with previously published work with canine
A\textsubscript{1}R and A\textsubscript{2A}R, where an
A\textsubscript{1}R chimera showed native coupling with
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i/o (Tucker et al., 2000).

In our previous work (Jain et al., 2018),
A\textsubscript{1}/A\textsubscript{2A}R chimera expression in yeast
showed exceptional yields of active receptors per cell as determined by
radioligand binding (B\textsubscript{max}), as compared to wild-type
A\textsubscript{1}R. The dissociation constant for NECA for the
A\textsubscript{1}/A\textsubscript{2A}R was similar to the reported
values for A\textsubscript{1}R (14 nM in (Stewart et al., 2009)),
suggesting native-like affinity. Here, we explored whether the chimera
showed similar efficacy in downstream signaling as compared to wild-type
A\textsubscript{1}R receptor in yeast. The dose-dependent mCherry
fluorescence response for each receptor was comparable when the
non-specific agonist, NECA, was added (Figure 6E).

To test the role of the A\textsubscript{2A}R C-terminus on
A\textsubscript{1}R signaling in mammalian cells, cAMP was measured in
transiently transfected HEK-293 cells. The addition of the
A\textsubscript{2A}R C-terminus to A\textsubscript{1}R did not lead to
constitutive activity of the receptor in the absence of ligand (Figure
7, blue open bars); therefore, 10 \selectlanguage{greek}μ\selectlanguage{english}M forskolin was used to enable a
basal cAMP signal. When treated with the A\textsubscript{1}R-specific
agonist CPA (1 \selectlanguage{greek}µ\selectlanguage{english}M, in the presence of 10 \selectlanguage{greek}μ\selectlanguage{english}M forskolin), cells
transfected with either A\textsubscript{1}/A\textsubscript{2A}R or
A\textsubscript{1}/A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R showed a moderate reduction
in cAMP signaling (72\selectlanguage{ngerman}±10\% and 67±10\%, respectively) compared to
forskolin treatment, consistent with the wild-type A\textsubscript{1}R
(62±3\%), verifying that A\textsubscript{1}/A\textsubscript{2A}R and
A\textsubscript{1}/A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R chimeras couple to
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i (Figure 7, green hatched bars). When treated with
the A\textsubscript{2A}R-specific agonist CGS21680 (1 \selectlanguage{greek}μ\selectlanguage{english}M, in the
presence of 10 \selectlanguage{greek}μ\selectlanguage{english}M forskolin) cells transfected with either
A\textsubscript{1}/A\textsubscript{2A}R or
A\textsubscript{1}/A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R showed a negligible change
in cAMP signaling compared to forskolin treatment alone, verifying that
A\textsubscript{1}/A\textsubscript{2A}R chimeras do not bind
A\textsubscript{2A}R-selective agonist or couple to G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s
(Figure 7, blue solid bars). The results obtained are consistent with
our observations in yeast. Taken together, these results suggest that
the C-terminus of the adenosine receptors does not play a role in the
specificity of the GPCR-G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}interaction but is necessary
to produce a downstream signaling response.

\emph{3.4 Dimerization of A\textsubscript{2A}R is not necessary for the
downstream signaling response}

The C-terminus of A\textsubscript{2A}R has been hypothesized to be
involved in oligomerization of the receptor (Navarro et al., 2018;
Schonenbach et al., 2016). Thus, truncation of A\textsubscript{2A}R
could result in loss of oligomerization, leading to the observed lack of
downstream signaling. To test this possibility, we measured the
downstream signaling response of three A\textsubscript{2A}R variants,
S374A, C394S, and C394A, that are located on the A\textsubscript{2A}R
C-terminus and have been shown previously to disrupt oligomerization
(Borroto-Escuela et al., 2010; Schonenbach, 2017; Schonenbach et al.,
2016). The A\textsubscript{2A}R S374A variant was shown to be incapable
of forming A\textsubscript{2A}R-Dopamine D\textsubscript{2}R oligomers
and to abolish A\textsubscript{2A}R-mediated inhibition of
D\textsubscript{2}R signaling (Borroto-Escuela et al., 2010).
Schonenbach and colleagues showed that a cysteine mutation at residue
394 led to a loss of dimer and higher oligomer formation in purified
A\textsubscript{2A}R protein variants (Schonenbach et al., 2016). Here,
the variants showed expression levels comparable to the wild-type
receptor as measured by whole cell fluorescence of mCitrine-tagged
receptor (data not shown). After the addition of 100 \selectlanguage{greek}μ\selectlanguage{english}M NECA, all the
variants showed downstream signaling similar to wildtype
A\textsubscript{2A}R in yeast (Figure 8). Furthermore the C394S and
C394A variants had no apparent change in EC50 values as compared to the
wild-type receptor (data not shown). Thus, an inability to oligomerize
did not affect the signaling activity.

\textbf{4. Discussion}

Since the early nineties, the engineered yeast MAPK response pathway has
been known as a useful tool to study human GPCR signaling and identify
lead drug candidates by recapitulating native dose-response binding
preferences (Fowlkes et al., 1997; King et al., 1990; Stewart et al.,
2009). Both A\textsubscript{1}R and A\textsubscript{2A}R have been shown
previously to interact with yeast/human chimeric
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}protein to produce downstream signaling responses in
the engineered yeast (Bertheleme et al., 2013; Knight et al., 2016;
Peeters et al., 2012; Stewart et al., 2009). Here, engineered yeast
strains from different parental backgrounds successfully captured
A\textsubscript{1}R and A\textsubscript{2A}R downstream signaling via
their corresponding native G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}proteins. Strains obtained
from the Broach laboratory showed a higher signal to noise ratio than
those from the Dowell laboratory under these conditions, indicating that
other parental strain differences can impact the signal-to-noise
obtained in cell-based assays.

One of the strengths of the engineered yeast is the capability of
quantifying the GPCR- G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}interaction at a common
endpoint of the signaling cascade. This allows direct comparison of the
strengths of the activation for different G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}biased
ligands. One such study performed by Stewart et al. (2009) identified a
novel A\textsubscript{1}R agonist with biased specificity for
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i vs G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}o coupling. Efforts have been
made to replicate this model of utilizing the last five amino acids of
the C-terminus of the G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}protein into a mammalian system
using G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s or G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}q as templates (Conklin
et al., 1993; Hsu et al., 2007). A study by Hsu and Lou (2007)
implementing this approach in HEK-293 cells tested the interaction of
A\textsubscript{1}R with G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s chimeras via a cAMP assay.
The authors observed cAMP production for all G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}variants
tested except G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s, suggesting the system was not
effective in capturing the specificity of the interaction of
A\textsubscript{1}R with G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}proteins. In contrast, our
results with native-like G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}coupling may have resulted
from higher than native levels of G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}protein expressed
(\textasciitilde{}three-fold higher than mock transfected, native HEK)
(Geppetti et al., 2015; Kostenis et al., 2005) and because additional
GPCRs present in the HEK cells led to signal promiscuity (O'Hayre et
al., 2013). In our study, all the signaling components in the engineered
yeast were expressed under their native promoters and perhaps as a
result, the yeast cell assay more effectively captured the specificity
of GPCR-G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}interaction.

The long C-terminus of the A\textsubscript{2A}R (122 amino acids) is
assumed to be highly flexible and disordered; thus, crystallization of
adenosine receptors has all focused on using truncated receptors. Here,
we tested the ability of truncated receptors to couple with
G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}protein to produce downstream signaling. Both for
A\textsubscript{1}R and A\textsubscript{2A}R, a C-terminal truncation
resulted in no downstream signaling. These observations in yeast were
validated in transiently transfected mammalian cells. For
A\textsubscript{1}R, our observations are consistent with those of
Pankevych et al (2003), who observed that the truncated rat
A\textsubscript{1}R receptor variants showed inefficient trafficking to
the plasma membrane, reduced ligand binding, and downstream signaling,
depending on the length of the C-terminus. We observed similar
inefficient localization and loss of downstream signaling for the human
A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R.

Here we find that the A\textsubscript{2A}R C-terminus did not change the
G-protein coupling preference from G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i to
G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s for the A\textsubscript{1}/A\textsubscript{2A}R
variants. Our results were consistent with previously published work by
Tucker et al. (2000) that found that a chimera of canine
A\textsubscript{1}R with a canine A\textsubscript{2A}R C-terminus showed
no change in G-protein coupling behavior. The human
A\textsubscript{1}/A\textsubscript{2A}R chimera showed a dose-dependent
fluorescent response similar to the wild-type receptor, suggesting there
was no change in ligand binding or G-protein coupling behavior due to
the presence of the A\textsubscript{2A}R C-terminus. Taken together with
our previous results of exceptional yields of the chimera (Jain et al.,
2018), these data suggest that A\textsubscript{1}/A\textsubscript{2A}R
could be an effective variant to study biophysical characteristics and
ligand binding for the A\textsubscript{1} receptor.

The A\textsubscript{2A}R C-terminus is known to interact with many
accessory proteins in the GPCR signaling pathways like G protein
receptor kinases and \selectlanguage{greek}β-\selectlanguage{english}arrestins that aid in receptor signaling and
desensitization (Gsandtner et al., 2006; Keuerleber et al., 2011; Zezula
et al., 2008) but, has previously been thought to be dispensable for
G-protein signaling (Klinger et al., 2002; Palmer et al., 1997). Bennett
et al (2013) showed that A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R expressed by an
inducible promoter was capable of coupling to G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s in a
receptor expression-level dependent manner; however, their data was
normalized, and total cAMP levels not reported. We do see a small
increase in ligand-dependent signaling for the A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R
truncation (Fig 4A), but the signal is over twenty fold less than wild
type A\textsubscript{2A}R, suggesting the truncation is responsible for
the loss of G protein signaling.

The A\textsubscript{2A}R receptor has been shown to form homo-oligomers
in native, mammalian systems, and in yeast (Canals et al., 2004; Ciruela
et al., 2011; Ferre et al., 2007; McNeely, 2016; Vidi et al., 2008). The
long C-terminus of the A\textsubscript{2A}R has been shown to interact
with dopamine receptors and has been hypothesized to be involved in
homo- oligomerization, which may impact signaling (Ciruela et al., 2011;
Schonenbach, 2017). Our results show that the monomeric
A\textsubscript{2A}R variants were still capable of native-like
downstream signaling. Taken together, our results highlight the role of
the C-terminus for A\textsubscript{2A}R and A\textsubscript{1}R in
G-protein coupling, but not in G-protein specificity.

\textbf{5. Acknowledgements}

The authors thank Prof. James Broach for providing the CY13393 and
CY13399 yeast strains and Dr. Simon Dowell (Glaxo Smith Kline, UK) for
providing Gpa1-human G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}chimera yeast strains.



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\textbf{Figure legends}

\textbf{Figure 1.} A) GPCR-mediated MAPK signaling cascade in yeast. In
this engineered pheromone response signaling pathway, cells express a
yeast/human chimeric G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}protein to enable human GPCRs to
couple with the yeast signaling pathway. Upon activation of downstream
signaling, cells express mCherry fluorescent protein, which acts as an
indirect measure of receptor activation. B) Schematic represents
approach to homologous recombination to include the mCherry gene along
with the antibiotic resistance gene for clone selection in the Fus1
locus in yeast strains. C) Agonist (100 \selectlanguage{greek}μ\selectlanguage{english}M NECA, green hatched bars)
mediated downstream signaling responses as compared to control (DMSO,
red closed bars) for A\textsubscript{1}R were measured in cells modified
pheromone response pathway in yeast expressing Gpa1p-human
G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}chimeras in GSK strains (A; mean \selectlanguage{ngerman}± S.D., for three
independent experiments).

\textbf{Figure 2.} Agonist (100 \selectlanguage{greek}μ\selectlanguage{english}M NECA, green hatched bars) mediated
downstream signaling responses as compared to control (DMSO, red closed
bars) for A\textsubscript{1}R (A) and A\textsubscript{2A}R (B) were
measured in cells modified pheromone response pathway in yeast
expressing Gpa1p-human G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}chimeras in Broach laboratory
strains (mean \selectlanguage{ngerman}± 95\% C.I., n=6, 3 independent transformants performed in
duplicate).

\textbf{Figure 3.} Absence of the C-terminus for A\textsubscript{1}R and
A\textsubscript{2A}R resulted in a loss of the signaling response in
yeast strains. A) Full-length expression of mCitrine tagged wild-type
and truncated A\textsubscript{1}R and A\textsubscript{2A}R was observed
using Western blot analysis with an anti-GFP antibody. Full-length
receptor is indicated by an arrow. Molecular weights were estimated
using Precision Plus Protein Western C standards. The expected molecular
weight of each receptor is as follows: A\textsubscript{1}R, 63.4 kDa;
A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R, 59.1 kDa; A\textsubscript{2A}R, 71.6 kDa; and
A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R, 66.1 kDa. B) Representative confocal images
yeast cells expressing i) A\textsubscript{1}R, ii) A\textsubscript{2A}R,
iii) A\textsubscript{1}\selectlanguage{greek}Δ\selectlanguage{english}291R and iv) A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R show
membrane trafficking of the receptor, as indicated. A\textsubscript{2A}R
and its truncated receptor showed efficient trafficking to the plasma
membrane whereas A\textsubscript{1}R and its truncation showed
intracellular puncta with some receptor localized at the membrane. MAP
kinase response signaling of the full length and truncated receptor in
(C) Gpa1p-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i1(5) and (D) Gpa1p-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s (5)
strains. NECA (100 \selectlanguage{greek}μ\selectlanguage{english}M) shown as green hatched bars and DMSO as red bars.
Data represents the mean \selectlanguage{ngerman}± 95\% C.I. for experiments performed in
duplicate for three independent transformants. Note that the signaling
response of A\textsubscript{1}R and A\textsubscript{2A}R from Figure 2
are replotted in Fig C and D to facilitate comparison of the truncations
to the full-length receptors across each G-protein chimera.

\textbf{Figure 4.} Transiently transfected HEK-293 cells were used to
determine downstream signaling for chimeras. A) Agonist-mediated cAMP
accumulation for transiently transfected cells with A\textsubscript{2A}R
and A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R (no ligand shown in red filled bars, 1 \selectlanguage{greek}μ\selectlanguage{english}M
CGS21680 in green hatched bars). Data represent mean \selectlanguage{ngerman}± S.E.M. for three
independent transfections performed in triplicate (*p\textless{}0.001,
Student's t-test). B) Western blot analysis of A\textsubscript{2A}R and
its truncation from transiently transfected in HEK-293, as obtained from
total cell lysate or membrane fractions. Precision Plus Protein Western
C standards were used to determine molecular weight as indicated. Dimer
and full-length receptor are indicated by an arrow for
A\textsubscript{2A}R. The A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R shows a smaller band
visible at \textasciitilde{}30 kDa that is likely a proteolytic product,
also indicated by an arrow. Expected molecular weights for
A\textsubscript{2A}R is 44.7 kDa and A\textsubscript{2A}\selectlanguage{greek}Δ\selectlanguage{english}316R is 35.1
kDa, and molecular weight markers were estimated using Precision Plus
Protein Western C standards.

\textbf{Figure 5.} Inhibition of cAMP production after forskolin
stimulation in transiently transfected HEK-293 cells with full-length
A\textsubscript{1}R as compared to truncated A\textsubscript{1}R (no
ligand in blue open bars, 10 \selectlanguage{greek}μ\selectlanguage{english}M forskolin in red filled bars and 10 \selectlanguage{greek}μ\selectlanguage{english}M
forskolin and 1 \selectlanguage{greek}μ\selectlanguage{english}M CPA in green hatched bars). Data represents mean \selectlanguage{ngerman}±
S.E.M. for three independent transfections performed in triplicate
(*p\textless{}0.001, Student's t-test).

\textbf{Figure 6.} Expression and downstream MAPK signaling response in
yeast for A\textsubscript{1}/A\textsubscript{2A}R chimera with
full-length and truncated C-terminus show native
A\textsubscript{1}R-like behavior. A) Western blot images showing
expression of mCitrine tagged receptors for full-length and truncated
chimeric receptors. Precision Plus Protein Western C standards were used
to determine molecular weight as indicated. B) Representative confocal
images of yeast strains showing receptor localization of full length and
truncated A\textsubscript{1}/A\textsubscript{2A}R chimera. Both the
full-length and truncated A\textsubscript{1}/A\textsubscript{2A}R
chimera produce signaling response in inhibitory
Gpa1p-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}i1(5) strain (C), but not in stimulatory
Gpa1p-G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}s(5) strains (D). (E) Dose-response curve for
A\textsubscript{1}/A\textsubscript{2A}R chimera (blue squares) is
similar to the native A\textsubscript{1}R receptor (red circle). 100 \selectlanguage{greek}μ\selectlanguage{english}M
NECA is shown as green hatched bars and DMSO in red filled bars. The
signaling response for A\textsubscript{1}R is replotted from Figure 2.
Data represents the mean \selectlanguage{ngerman}± 95\% C.I. for experiments performed in
duplicate for three independent transformants.

\textbf{Figure 7.} Inhibition of cAMP production after forskolin
stimulation in HEK-293 cells transiently transfected with full length
and truncated A\textsubscript{1}R (no ligand in blue open bars, 10 \selectlanguage{greek}μ\selectlanguage{english}M
forskolin in red filled bars, 10 \selectlanguage{greek}μ\selectlanguage{english}M forskolin and 1 \selectlanguage{greek}μ\selectlanguage{english}M CPA in green
hatched bars and 10 \selectlanguage{greek}μ\selectlanguage{english}M forskolin and 1 \selectlanguage{greek}μ\selectlanguage{english}M CGS21680 in blue filled bars).
The signaling response for A\textsubscript{1}R is replotted from Figure
5. Data represents the mean \selectlanguage{ngerman}± S.E.M. for three independent transfections
performed in triplicate (*p\textless{}0.001 and \#p\textless{}0.01,
Student's t-test).

\textbf{Figure 8.} Dimerization of A\textsubscript{2A}R is not required
for signaling. Three A\textsubscript{2A}R variants -- S374A, C394A and
C394S -- previously reported to be incapable of dimer formation give
MAPK signaling in yeast comparable to wild-type A\textsubscript{2A}R
receptor. 100 \selectlanguage{greek}μ\selectlanguage{english}M NECA is shown in green hatched bars and DMSO (control)
in red filled bars. The signaling response for A\textsubscript{2A}R is
replotted from Figure 2. Data represent mean \selectlanguage{ngerman}± 95\% C.I. for experiments
performed in duplicate for three independent transformants.\selectlanguage{english}
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