Associate editor:J.Wess
Orphan GPCRs and their ligands
Olivier Civelli *,Yumiko Saito,Zhiwei Wang,Hans-Peter Nothacker,Rainer K.Reinscheid
Department of Pharmacology and Department of Developmental and Cell Biology,University of California,Irvine,
Med Surge II Room 369,Irvine,CA 92697-4625,USA
Abstract
Due to their diversity,G-protein-coupled receptors (GPCRs)are major regulators of intercellular interactions.They exert their actions by being activated by a vast array of natural ligands,referred to in this article as ‘‘transmitters’’.Yet each GPCR is highly lective in its ligand recognition.Traditionally,the transmitters were found first and rved to characterize the receptors pharmacologically.Since the end of the 1980s,however,it is the GPCRs that are first to be found becau they are identified molecularly by homology screening approaches.But the GPCRs found this way suffer of one drawback,they lack their natural transmitters,they are ‘‘orphan’’GPCRs.Searchi
ng for transmitters of orphan GPCRs has given birth to the rever pharmacology approach that us orphan GPCRs as targets to identify their transmitters.The most salient success of the rever pharmacology approach were the discoveries of 9novel neuropeptide families.The have enriched our understanding of veral important behavioral respons.But the application of rever pharmacology has also led to some surprising results that question some basic pharmacological concepts.This review aims at describing the history of the orphan GPCRs and their impact on our understanding of biology.D 2005Elvier Inc.All rights rerved.
Keywords:Orphan GPCR;Rever pharmacology;Transmitters;Neuropeptides
Contents
1.
<5251.1.The transmitters .....................................5261.2.The physiological importance of the G-protein-coupled receptors ...........5262.The orphan G-protein-coupled receptors .................
...........5262.1.Rever pharmacology .................................5272.2.The arch for novel transmitters .
...........................5272.3.Physiological roles of the orphan G-protein-coupled receptors .............5283.The surpris of the rever
...........5283.1.The 5293.2.The promiscuous G-protein-coupled receptors .....................5293.3.The non-lective G-protein-coupled receptors .....................5294.The impact of the orphan G-protein-coupled
530
1.Introduction
The minal discovery that the h 2-adrenergic receptor and the opsins share a 7-transmembrane domains (TMs)topology (Dixon et al.,1986),created the concept that receptors that
0163-7258/$-e front matter D 2005Elvier Inc.All rights rerved.doi:10.1016/j.pharmthera.2005.10.001
*Corresponding author.Tel.:9498242522;fax:9498244855.E-mail address: ocivelli@uci.edu (O. Civelli).
Pharmacology &Therapeutics 110(2006)525–
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532
/locate/pharmthera
伤心的微信名couple to G proteins form a homologous supergene family, thereafter called the G-protein-coupled receptors(GPCRs) (Hall,1987).The7TMs topology has since evolved to include receptors that do not couple to G proteins,and our under-standing of receptor activation has been shown to include proteins other than the G proteins(Bockaert&Pin,1999; Angers et al.,2002;Pierce et al.,2002;Kristiann,2004).Yet, one tenet of the basic concept has remained constant over the last20
years:all the small molecules that have evolved to direct intercellular interactions interact with GPCRs(7TMRs).This positions the GPCRs at the center of signal transmission and endows them with an extraordinary importance in the organism’s life and survival.
1.1.The transmitters
The GPCRs are activated by a plethora of‘‘transmitters’’(Civelli et al.,2001),the first mesngers that are either prent in the environment or relead from a cell to carry a message to a cond one.The act in an endocrine,paracrine,or exocrine fashion to allow the organism to react to particular physiolog-ical challenges.Transmitters are mostly small molecules although few of them are larger polypeptides.They include biogenic amines,neuropeptides,chemokines,lipid mediators, nucleotides,amino acid and derivatives,polypeptide hor-mones,pheromones,olfactory and gustatory molecules,and some other naturally occurring chemicals such as calcium ions and protons.
Traditionally,the GPCRs are expected to exhibit specificity for the transmitters(Goldstein,1974).This specificity results from evolutionary process that aim at diversifying the intercellular interactions.A transmitter may be the natural ligand of more than one GPCR,but tho then share a higher degree of homology that groups them into a subfamily.Also a GPCR may bind more than one transmitter,but
then the transmitters share structural similarities and are often part of the same synthesis pathway,as in the ca of the neuropeptides synthesized from the same precursor(Douglass et al.,1984).A classic example for structurally similar ligands binding structurally related receptors are the opioid receptors and the natural opioid peptides.The concept of GPCR specificity has recently been revid and will be discusd in this review. 1.2.The physiological
importance of the G-protein-coupled receptors
Becau they direct such a broad spectrum of interactions, the GPCRs participate in about every physiological respon. Each cell express a few dozen different GPCRs,which implies that its homeostasis can be influenced by numerous transmitters.GPCR activation leads to intracellular chemical changes that may affect directly the state of the cells but can also lead to transcriptional regulation,thus making the role of the GPCRs long lasting.Conquently,every function in the organism can be affected by the activities of particular GPCRs.
For example,the cardiovascular,respiratory,muscular,and endocrine functions are directly dependent on the activities of the adrenergic,histaminergic,muscarinic,and hormone GPCRs,respectively.The organ that is possibly most dependent on GPCR activities is the brain,where practically all the GPCR曼彻斯特大学
s are expresd and where their activities add a slower but long lasting dimension to the synaptic transmission mechanisms that direct all brain-related respons.In addition, one will remember that most of our ns such as olfaction, taste,and vision depend directly on the activation of specific GPCRs.
A particular GPCR is often expresd in veral tissues.It can be found in the periphery and in the CNS.Its roles in the tissues may ultimately be very different although the cond mesngers that result from its initial activation are probably the same.We only begin now to disct the role that the activation of a particular GPCR has on the organism.In some cas,we are helped by the studies of some pathological cas (Scho¨neberg et al.,2004).Furthermore,and increasingly during the past few years,we expect that genome quence analys will point at GPCR structural differences(Balasu-bramanian et al.,2005)that may help explain pathological differences and conquently unravel the roles of particular GPCRs in particular tissues.But ultimately,the discovery of the predominant role(s)of a GPCR awaits the development of corresponding surrogate agonist or antagonist molecules.Only administration of such drugs to human can answer the question of the GPCR function.Fortunately,developing specific surrogate ligands for GPCRs is in the realm of medicinal chemistry.Indeed about half of the drugs prently on the market are targeting GPCRs(Dre
ws,2000)and studies of their effects has been a driving force in our understanding of the functions of the GPCRs.Yet the are far from covering the entire supergene family,which amount to some800receptors in the human genome(Vassilatis et al.,2003).
This review will describe how the GPCRs became such an imposing supergene family and how they continue to spear-head our understanding of particular physiological respons.It will focus on the arch for the ligands of the transmitter GPCRs and will proceed from specificity,to diversity,to finally complexity.
2.The orphan G-protein-coupled receptors
Most of the GPCRs started as orphans.Their discoveries stemmed from the concept that GPCRs would belong to a supergene family and thus would share quence similarities. Homology screening techniques,low stringency hybridization (Bunzow et al.,1988,1992)soon followed by PCR-derived approaches(Libert et al.,1989),paved the way for the discoveries of new GPCRs,thus that,by the end of the 1980s,it became clear that the number of GPCRs would be large.This was confirmed,at the turn of the century,with the quence of the genome.Today,one estimates that the number of GPCRs is about800,of which more than half are olfactory GPCRs(Vassilatis et al.,2003).
The discovery of new GPCRs found by homology screening suffers from one obvious problem,the receptors found lack their pharmacological identities,their natural ligands.They are
O.Civelli et al./Pharmacology&Therapeutics110(2006)525–532 526
all‘‘orphan’’receptors(Libert et al.,1991a,1991b).The pursuit to unravel their identities was thought to be formidable and to many an unresolvable task that would lead to unglamorous fishing expeditions.
2.1.Rever pharmacology
In the cond part of the1980s,there existed about50 transmitters,potential GPCR ligands,that had no cloned cognate receptors.Testing all of the for their binding to new GPCRs was en as an impossible aim in an academic environment.Yet rendipity and ingenious insights,such as the that could spring from the analysis of the new GPCR tissue expression profile,proved to be successful in matching the first orphan GPCRs to known transmitters.The first deorphanized GPCRs,the5HT-1A and the D2dopamine receptors,were already reported in1988(Fargin et al.,1988; Bunzow et al.,1988).The strategies ud were the same,that is,the orphan GPCR was expresd by DNA transfection in eukaryotic cells,membranes of the cells were then ud as targets to determine the binding of potential transmitters.This strategy has become to be known as rever ph
armacology (Libert et al.,1991a,1991b;Mills&Duggan,1994).
During the first part of the1990s,application of the rever pharmacology strategy led to the pharmacological character-ization of many GPCRs.The endeavors were mostly carried out in an‘‘artisanal’’fashion;the focus was on one particular receptor tested for few potential known ligands(Table1).At the same time,however,random arches for new GPCRs, using PCR-bad homology screening approaches,were also in full swing,thus the overall number of orphan GPCRs was steadily increasing(Marche et al.,1994,1998).This led us to conclude that the GPCRs outnumbered the known potential ligands and more importantly that the receptors must bind ligands that have not been thus far characterized becau inactive receptors should be evolutionarily discarded(Civelli, 1998).This recognition inspired enough confidence in a few rearchers to utilize orphan GPCRs as baits to isolate their natural ligands,which meant to identify novel transmitters. 2.2.The arch for novel transmitters
The concept that an orphan receptor could be ud as bait to identify a novel transmitter requested the application of two technologies(Civelli,1998).First,it necessitated the arch for novel transmitters in tissue extracts,therefore in complex molecular mixtures.Then it necessitated the monitoring of receptor activation instead of binding.Both requirements implied the application of coun
tless repetitive assays.Fortu-nately,such assays were existing or being developed for the
pharmaceutical industry(Wess et al.,2001).
The cloning of receptors,enzymes,and other targets of pharmaceutical interest had revolutionized drug screening (Drews,2000).From that time on,drugs could be arched for by the random screening of large libraries of synthetic compounds(Schreiber,2000).The are tested for the desired biological and in vivo activity and,if active,chemically modified to fulfill the pharmaceutical constraints of expected drugs.The screening of libraries of compounds required high-throughput assays and such assays were developed with the aim of monitoring GPCR reactivity.
The first orphan GPCR that was ud for discovering a novel transmitter was ORL-1,cloned through its homology to the opioid receptors(Henderson&McKnight,1997).Its Table1
Dates in the history of GPCR deorphanization
1986The h2-adrenergic and the rhodopsin receptors share a
7-transmembrane topology.
Birth of the GPCR family concept and recognition that
homology screening approaches could lead to new
GPCRs.
1987Identification of G-21,the first orphan GPCR using low
stringency hybridization.
1988The first deorphanizations:G-21is recognized as the
5-HT1A receptor,RGB-2as the dopamine D2receptor.
Introduction of the rever pharmacology approach.
1989Development of the PCR-bad homology screening
approach.
1990–1995Orphan GPCRs are identified by homology screening.
The‘‘artisanal’’era of GPCR deorphanization.
The following GPCRs are characterized:Adenosine A1,
A2a,A2b,A3;adrenergic a1A,a1d,a2b,a2c,b1,b3;
anaphylatoxin C3a,C5a;angiotensin AT1b;bombesin
BB1,BB3;cannabinoid CB1,CB2;chemokine CCR1,
CCR2,CCR3,CCr4,CCR5,CXCR2,CXCR3,CXCR4;
cholecystokinin CCKa;dopamine D1,D2,D3,D4,D5;
follicle-stimulating hormone;formyl-peptide FPR2;
galanin type2;gonadotropin-releasing hormone;
histamine H2;lysophosphatidic acid;melanocortin MC1,
MC2,MC3,MC4,MC5;melatonin ML1a,ML1B;
muscarinic acetylcholine M3,M4,M5;neurokinin NK1,
NK3;neuropeptide YY1,YY1-like,YY4;neurotensin
NTR2;opioid and A;prostanoid EP1,EP2,EP3,EP4,DP,
FP,IP;protea-activated2,3;purinoceptor P2Y1,P2Y3,
P2Y4,P2Y6,P2Y8;rotonin5-HT1a,5-HT1b,5-HT1d,
5-HT1e,5-HT1f,5-HT2a,5-HT2b,5-HT4,5-HT5a,
5-HT5b,5-HT6,5-HT7;somatostatin SST1,SST2,SST3,
SST4,SST5;thyrotropin-stimulating hormone;
vasopressin V1b,V2.
1995Discovery of the first novel natural ligand of an orphan
GPCR:nociceptin/orphanin FQ.Demonstration that
orphan GPCRs can be ud to identify new transmitters. 1996–2005The‘‘industrial’’era of GPCR deorphanization.
The following GPCRs are identified:leukotriene B4,C4,
D4;latrotoxin;sphingosine1-phosphate;lysophosphatidic
acid;melanin-concentrating hormone;urotensin II;motilin;
neuromedin U;UDP-gluco;sphingosylphosphorylcholine;
histamine3;prostaglandin D2;neuropeptide FF,AF;
RFamide-related protein1,3;lysophosphatidylcholine;
adenosine diphosphate;psychosine;trace amines;
5-oxo-ETE;bile acids;bovine adrenal medulla peptide22;
relaxin;relaxin-3;bradykinin;pyroglutamylated
arginine-phenylalanine-amide peptide;cortistatin;medium
and long fatty acids;nicotinic acid;proton;h-alanine;
a-ketoglutarate.美容心得
1998–2004Discovery of8novel neuropeptide/receptor system:
Orexins/hypocretins,prolactin-releasing peptide,apelin,
ghrelin,metastatin,neuropeptides B/W,prokineticins1/2,
neuropeptide S.
The dates prented in this table refer to the mentioned in text.The GPCR lists were derived from Marche et al.(1998)and Saito and Civelli (2005).
O.Civelli et al./Pharmacology&Therapeutics110(2006)525–532527
activation was monitored by quantifying intracellular decreas in cAMP levels,which could be measured in newly developed scintillation proximity assays.Becau phylogenic analys classified ORL-1as a peptidergic GPCR and becau ORL-1is expresd in the CNS,peptidergic brain tissue extracts were prepared,purified,and fractionated.Fractions were tested for their abilities to inhibit adenylyl cycla activity in cells that were stably transfected with ORL-1.A17-residue long peptide w
as ultimately isolated,named orphanin FQ or nociceptin (OFQ/N)(Meunier et al.,1995;Reinscheid et al.,1995).Its structural similarities to the opioid peptides immediately attracted considerable attention,yet it has been proven not to bind the opioid receptors(Reinscheid et al.,1998).
The cond successful attempt at discovering novel trans-mitter through orphan GPCRs screened over50different orphan GPCRs by measuring their abilities to induce intracel-lular calcium relea when subjected to peptidic extracts.One receptor did respond and led to the characterization of two peptides,the orexins(Oxs)(Sakurai et al.,1998),also identified through an RNA subtraction approach as hypocretins (Hcrts)(de Lecea et al.,1998).This was immediately followed by the discovery of two novel peptides,prolactin-releasing peptide,and apelin as the natural ligands of the orphan GPCRs GPR10and APJ,respectively(Hinuma et al.,1998;Tatemoto et al.,1998).
The success proved the validity of high-throughput screening of orphan GPCRs.It is therefore not surprising that the pharmaceutical industry became its major proponent (Hinuma et al.,1999).Conquently,orphan GPCRs began to be screened randomly against large libraries of ligands, tting the stage for the‘‘industrial’’period of deorphanization (Wi et al.,2004).The libraries contained all the ligands that had not been matched to any receptor molecules but also many molecules that are known to exist in cells.This led in a few years to the deorphanization of som
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五行属火寓意最好的字e40GPCRs(Saito& Civelli,2005).
2.3.Physiological roles of the
orphan G-protein-coupled receptors
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The impact that the deorphanization of GPCRs had and continues to have on our understanding of the organism’s function is fundamental.Deorphanizing a GPCR opens the opportunity to combine anatomical studies on the site of synthesis with that on the sites of action of the system and thus to gain a full understanding of the localization of the system. Most often,the sites of the orphan GPCR expression rve as primary indication of the role of the system.The matching of orphan GPCRs to previously known transmitters has permitted such preci anatomical analys.Genetic ablations of the orphan GPCRs can also help to understand the new receptor systems(Morita et al.,2004;Piao et al.,2004).But the most striking results came from the discoveries of novel peptides. Our understanding of veral physiological respons has greatly gained from the discoveries.
Sleep is1physiological respon that has been impacted by the orphan GPCR rearch(Civelli,2005,in press).While GABA,noradrenalin,and histamine were known to regulate sleep,the discoveries of Hcrts/Oxs have proven that neuropep-tides have a prominent role.Inactivatio
n of one of the Hcrts/ Oxs receptor induces narcolepsy(Chemelli et al.,1999;Lin et al.,1999).More recently,3other orphan GPCR systems,the PrRP(Lin et al.,2002),neuropeptide S(Xu et al.,2004a, 2004b),and urotensin II(Huitron-Rendiz et al.,2005) systems have also been shown to regulate some aspects of sleep.Finally,the prokineticin system has been shown to be a major regulator of the circadian rhythm(Cheng et al.,2002).
Another physiological respon that has been shown to depend on the activity of orphan GPCRs is feeding(Xu et al., 2004a,2004b).While this respon was understood as relying on the relea of veral peptides,in particular leptin(Zhang et al.,1994),melanocyte-stimulating hormone(Marks&Cone, 2001),and neuropeptide Y(Gehlert,1999),it has been shown that the deorphanized GPCR systems of ghrelin(Kojima et al., 1999;Tschop et al.,2000),melanin-concentrating hormone (Shimada et al.,1998;Marsh et al.,2002),and the Oxs/Hcrts (Willie et al.,2001)play important roles in the central regulation of food intake.
Other physiological respons that have been found to be regulated by orphan GPCR systems include anxiety as it relates to stress,which is impacted by the activity of OFQ/N(Jenck et al.,1997;Koster et al.,1999)or NPS(Xu et al.,2004a,2004b).
It should be emphasized that,due their novelty,our understanding of the function of the novel neuropeptide systems is still at its infancy.Novel systems such as the are initially tested on the basis of receptor localization.Most of the orphan GPCRs are expresd in veral parts of the CNS, their activation should affect the behavioral respons related to the CNS centers.Conquently,the effects that administra-tion of the novel neuropeptide has on behavior depend on the assays ud.A novel neuropeptide may modulate a behavioral respon in a totally new fashion and that may lead to the discovery of a different function for that neuropeptide.The Hcrt/Ox effect on sleep is an example of such discovery (Chemelli et al.,1999;Lin et al.,1999).It is therefore likely that the deorphanized GPCRs may point at new behavioral or physiological respons that will enlarge our understanding of the function of the organism.
3.The surpris of the rever pharmacology approach
GPCRs have been deorphanized at a rate of7–8per year from1999until2004(Civelli,2005).This was mostly the result of large-scale random screenings of practically all molecules known to exist in cells.The primary pharmacolog-ical constraint of the screening endeavors was that active compounds exhibit affinities to the orphan GPCRs that are defined by the investigators.But there is no definite rule for predicting the affinity constant of a natural ligand at a particular receptor.Biogenic
amines,for example,activate their cognate receptors with potencies that are mostly in the micromolar range while most peptides do so in the nanomolar range. Moreover,the level of receptor expression in a transfected cell can affect ligand potency.Conquently,one has to remember
O.Civelli et al./Pharmacology&Therapeutics110(2006)525–532 528
that the rever pharmacology approach is bad on the investigator’s t standards and therefore subject to artefacts (i.e.,‘‘made by the art’’).
The screening endeavors took place mainly in pharma-ceutical companies.From a pharmaceutical standpoint,finding a compound that can activate a GPCR is the key to opening the door for the drug discovery process(Robas et al.,2003a, 2003b).It is not a prerequisite that this compound is the genuine transmitter.In doing so,many orphan GPCRs were matched to undoubtedly genuine transmitters,but some were matched to only surrogate ligands.For example,the orphan GPCR PUMA-G/HM74was matched to nicotinic acid(Soga et al.,2003;Tunaru et al.,2003),a success with therapeutic implications,but one that leaves open the door for the arch of the natural ligand.What grew from the inten arch for natural and surrogate ligands by rever pharmacology is that the number of potential genuine transmitters wound down.At that point,the technology began to be push
ed to its limits and transmitters began to be found that are unexpected.This is further compounded by some recent discoveries showing that some GPCRs are activated by veral transmitters that are chemically unrelated(Civelli,2005,in press).
3.1.The unexpected transmitters
An‘‘unexpected’’transmitter could best be defined as a naturally occurring molecule that was not expected to exert its action through a specific receptor.For example,UDP-gluco was found to activate the orphan GPCR KIAA0001with affinities in the100-nM range(Chambers et al.,2000).UDP-gluco was known to be a glucosyl donor in the biosynthesis of carbohydrates.Whether it acts as a transmitter has still to be shown.Other surprising examples are succinate and a-ketoglutarate,which are known as citric acid cycle intermedi-ates but were shown to activate the orphan GPCRs,GPR91 and99,respectively,with affinities in the25-to70-A M range (He et al.,2004).Succinic acid was identified not by random screening of defined ligands but through the purification of kidney extracts.Succinate was known to have a role on the reabsorption of phosphate and gluco in the proximal tubule and to stimulate gluconeogenesis.In GPR91-deficient mice, succinate was unable to induce hypertension,thus demonstrat-ing that its activity relies on GPR91activation(He et al.,2004). So there is no doubt that succinate is a genuine activator of GPR91,although the question remains w
hether it rves as a transmitter.Citric acid intermediates are not expected to be creted in a regulated manner.They may be relead upon mechanical stress or cell death,which would infer that some GPCRs are ud as monitors of metabolic breakdown or global injury.
3.2.The promiscuous G-protein-coupled receptors
Another issue regarding pharmacological lectivity has arin from some of the results of the application of rever pharmacology.Subfamilies of GPCRs usually bind one or veral cloly related ligands.Three opioid receptors all bind opioid peptides and have evolved their structure to insure that they do not bind OFQ/N(Reinscheid et al.,1998;Meng et al., 1998).Catecholamine receptors are structurally related as are their ligands.Yet the adrenergic and dopaminergic systems are viewed as parate,although it has been shown that adrenaline and noradrenalin can efficiently activate the dopamine D4 receptor(Lanau et al.,1997).But this does not hold for a recently discovered GPCR subfamily,the Mas-related GPCRs (Mrgs or nsory neuron-specific receptors;SNSRs).This is a family of orphan GPCRs that is predominantly expresd in dorsal root ganglions and thus might have a role in nociception.Variable numbers of Mrgs exist in human,rat, and mou making any attempt at orthologous classification difficult(Zylka et al.,2003).Being part of a subfamily,one could have expected that the Mrgs would bind similar transmitters.Instead,the Mrgs
have been paired to a variety of structurally diver transmitters:RFamide peptides for some mou Mrgs(Dong et al.,2001);BAM22(Lembo et al.,2002) and cortistatin(Robas et al.,2003a,2003b)for2human Mrgs; adenine(Bender et al.,2002)for a rat Mrg;and h-alanine (Shinohara et al.,2004)for an Mrg found in human,rat,and mou.The matched transmitters are specific to particular Mrgs and activate them efficiently.For example,the RFamides or BAM22peptides have affinities in the low nanomolar range (Lembo et al.,2002;Han et al.,2002),which is the range that is expected for peptides binding to GPCRs.So can it be that there are GPCR subfamilies that have a broad spectrum of transmitters and if so what does it imply for their function? From a physicochemical standpoint,if a molecule contains a motif that permits its interaction with a receptor,this interaction will take place.The issue is whether the receptor and that surrogate ligand will be in a position to interact in vivo.But this cannot be answered by rever pharmacology.
3.3.The non-lective G-protein-coupled receptors
Finally,when considering the outcomes of rever pharma-cology that go against one of the tenets of pharmacology,one has also to consider the ca of a receptor that is not lective in its ligand recognition.Such a receptor is the orphan GPCR GPRC6A.One has come to accept that glutamate,g
lycine,or GABA receptors do not bind other amino acids.GPRC6A,on the other hand,can be activated by a ries of basic l-a-amino acids with only a preference for basic amino acids(Well-endorph et al.,2005).For example,it is activated by l-Arg, with an affinity of about50A M.The concentration of l-Arg in the plasma is in the100-A M range.So,if Arg acts in an endocrine fashion,the receptor would be constantly and strongly activated.In addition,it would be surprising that any metabolic change would result in a high enough increa in the concentration of l-Arg to account for a significant difference in signaling.This suggests that l-Arg and the other GPRC6A ligands act in a paracrine fashion.Furthermore, becau GPRC6A belongs to the receptor family that include the calcium nsing receptor(Brown,1999),it may n free amino acid concentrations.Such a notion would help explain the promiscuity of GPRC6A in ligand recognition.
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