Phylogenetic analysis of 277 human G-protein-coupled receptors as a tool for the prediction of orphan receptor ligands.

BACKGROUND: G-protein-coupled receptors (GPCRs) are the largest and most diverse family of transmembrane receptors. They respond to a wide range of stimuli, including small peptides, lipid analogs, amino-acid derivatives, and sensory stimuli such as light, taste and odor, and transmit signals to the interior of the cell through interaction with heterotrimeric G proteins. A large number of putative GPCRs have no identified natural ligand. We hypothesized that a more complete knowledge of the phylogenetic relationship of these orphan receptors to receptors with known ligands could facilitate ligand identification, as related receptors often have ligands with similar structural features.
RESULTS: A database search excluding olfactory and gustatory receptors was used to compile a list of accession numbers and synonyms of 81 orphan and 196 human GPCRs with known ligands. Of these, 241 sequences belonging to the rhodopsin receptor-like family A were aligned and a tentative phylogenetic tree constructed by neighbor joining. This tree and local alignment tools were used to define 19 subgroups of family A small enough for more accurate maximum-likelihood analyses. The secretin receptor-like family B and metabotropic glutamate receptor-like family C were directly subjected to these methods.
CONCLUSIONS: Our trees show the overall relationship of 277 GPCRs with emphasis on orphan receptors. Support values are given for each branch. This approach may prove valuable for identification of the natural ligands of orphan receptors as their relation to receptors with known ligands becomes more evident.

Background

G-protein-coupled receptors (GPCRs) are the largest and most diverse family of transmembrane receptors. They respond to a wide range of stimuli including small peptides, lipid analogs, amino-acid derivatives, and sensory stimuli such as light, taste and odor [1], and transmit signals to the interior of the cell through interaction with heterotrimeric G proteins. Certain amino-acid residues of this receptor family are well conserved and approaches exploiting this, such as low-stringency hybridization and degenerate PCR, have been used to clone new members of this large superfamily [2,3,4]. Many of these putative receptors share GPCR structural motifs, but still lack a defined physiologically relevant ligand. One strategy to identify the natural ligand of these so-called orphan receptors uses changes in second-messenger activation in cells stably expressing the receptor in response to tissue extracts expected to contain the natural ligand [5]. In a second step, these extracts are tested and fractionated to purity, before being analyzed by mass spectrometry. This strategy led to the identification of several novel bioactive peptides or peptide families (for review see [6]). The identification of these natural ligands is likely to give further insight into the physiological role of these receptors and advance the design of pharmacologically active receptor agonists or antagonists. This is of particular interest, as GPCRs are the most targeted protein superfamily in pharmaceutical research [7]. Better prediction of the presumed chemical class or structure of the ligand facilitates the identification of orphan receptors by the strategy described above, as the ligand purification process can be tailored more specifically to the assumed class of substances.

Phylogenetic analysis of receptor relationships has already been used to elucidate the chemical nature of receptor ligands. The identification of sphingosine 1-phosphate as the ligand for the GPCR EDG-1 led to the prediction that EDG-3, EDG-5, EDG-6 and EDG-8 have the same ligand [8,9,10,11]. In contrast, phylogenetically distinct members of the EDG cluster - EDG-2, EDG-4 and EDG-7 - are receptors for the similar but distinct ligand lysophosphatidic acid (LPA) [12,13,14]. Neuromedin U, a potent neuropeptide that causes contraction of smooth muscle, was correctly predicted phylogenetically to be the ligand of the orphan GPCR FM3 (NMUR) [15]. Not only the ligand, but also the pharmacology of a novel receptor for histamine, was predicted and confirmed through phylogeny [16]. GPR86, related to the ADP receptor P2Y12, was similarly recently shown to bind ADP [17], and UDP-glucose, a molecule involved in carbohydrate biosynthesis, was shown to be the ligand for the related receptor KIAA0001 [18].

Mammalian GPCRs were previously classified by phylogeny into three families [19,20]: the rhodopsin receptor-like family (A), the secretin receptor-like receptor family (B) and the metabotropic glutamate receptor family (C). These results were generated by neighbor joining, a fast distance-based method suited for large datasets, but influenced by methodological flaws that can in part be overcome by methods not generally applied previously.

In this work, we compiled an exhaustive list that includes all available synonyms and accession numbers of 196 human GPCRs with known ligands and 84 human orphan receptors. The 241 sequences belonging to family A were aligned, and a tentative tree constructed by neighbor joining with 1,000 bootstrap steps. Subgroups of family A defined by this tree and sequences from families B and C were then used for more accurate phylogenetic analysis by state-of-the-art techniques. From this analysis, we tried to predict possible ligands for orphan receptors.

Results and discussion

We set out to define the phylogenetic relationship of human GPCRs by state-of-the-art tools, assuming that the identification of cognate ligands of orphan receptors will be facilitated by a more complete knowledge of their relationship within the large and diverse superfamily.

Database mining and multiple sequence alignment

Most receptors were identified by different groups; therefore, many confusing names and synonyms exist. We adhered to SWISS-PROT names where possible, and compiled a list including all available synonyms and accession numbers of 196 human GPCRs with known ligands and 84 human orphan receptors (Table 1 shows all receptors mentioned in this work; the complete list is supplied as an additional data file). Gustatory and olfactory receptors were omitted. Multiple protein sequences were aligned and the extremely variable amino termini upstream of the first transmembrane domain and carboxyl termini downstream of the seventh transmembrane domain were deleted to avoid length heterogeneity (see Figure 1). The deleted regions contained no significant sequence conservation.

Table 1

List of example receptor names, accession numbers and abbreviations

Receptor	Group	Accession no.	Names and synonyms
Human GPCR - Family A
ADMR	A02	O15218	Adrenomedullin receptor, Am-R
APJ	A03	P35414	Apelin receptor, Apj, Agtrl1
CML1	A08	Q99788	Chemokine receptor-like 1, Dez, Chemr23, Ch23, Cmklr1
CML2	A02	Q99527	Chemokine receptor-like 2, flow-induced endothelial G protein-coupled receptor, Feg-1, Gpr30, Cmkrl2, Dry12, Cepr
DUFF	A02	Q16570	Duffy antigen, Fy glycoprotein, glycoprotein D, Gpfy, Fy, Gpd, Darc
EDG1	A13	P21453	Endothelial differentiation, Sphingosine 1-phosphate receptor, Lp-B1
EDG2	A13	Q92633	Endothelial differentiation, lysophosphatidic acid receptor, Lp-A1, Vzg-1
EDG3	A13	Q99500	Endothelial differentiation, lysosphingolipid receptor, Lp-B3
EDG4	A13	NM_004720	Endothelial differentiation, lysophosphatidic acid receptor, Lp-A2
EDG5	A13	NP_004221	Endothelial differentiation, sphingolipid receptor, Lp-B2, H218, Agr16
EDG6	A13	AJ000479	Endothelial differentiation, lysosphingolipid receptor, Lp-C1
EDG7	A13	NP_036284	Endothelial differentiation, lysophosphatidic acid receptor, Lp-A3
EDG8	A13	NP_110387	Endothelial differentiation, sphingosine 1-phosphate receptor, Lp-B4
ETBR-LP2	A07	Y16280	Endothelin B receptor-like protein-2, Etbrlp2, Ebp2, Cns2
FSHR	A10	P23945	Follicle stimulating hormone receptor, Fsh-R, follitropin receptor
GPR	A06	NM_007223	G protein-coupled receptor
GPR1	A08	P46091	G protein-coupled receptor Gpr1
GPR3	A13	P46089	G protein-coupled receptor, Acca orphan receptor
GPR6	A13	P46095	G protein-coupled receptor 6
GPR7	A04	P48145	G protein-coupled receptor 7
GPR8	A04	P48146	G protein-coupled receptor 8
GPR25	A03	NM_005298	G protein-coupled receptor 25
GPR27	A18	NM_018971	G protein-coupled receptor 27, Sreb1
GPR34	A12	NM_005300	G protein-coupled receptor, Gpry
GPR35	A15	NM_005301	G protein-coupled receptor 35
GPR37	A07	NM_005302	G protein-coupled receptor 37, Endothelin receptor type B-like, Cns1
GPR39	A07	O43194	G protein-coupled receptor Gpr39
GPR40	A11	O14842	G protein-coupled receptor Gpr40
GPR41	A11	O14843	G protein-coupled receptor Gpr41, Hia-R
GPR42	A11	O15529	G protein-coupled receptor Gpr42
GPR43	A11	O15552	G protein-coupled receptor Gpr43
GPR44	A08	AAD21055	G protein-coupled receptor 44
GPR44	A08	AAD21055	G protein-coupled receptor 44
GPR48	A10	NM_018490	G protein-coupled receptor 48
GPR49	A10	NM_003667	G protein-coupled receptor 49, Hg38, G protein-coupled receptor 67, Fex
GPR52	A18	Q9Y2T5	G protein-coupled receptor Gpr52
GPR55	A15	NM_005683	G protein-coupled receptor 55
GPR57	A17	NM_014627	G protein-coupled receptor 57
GPR58	A17	NM_014626	G protein-coupled receptor 58
GPR61	A18	AF317652	G protein-coupled receptor 61
GPR62	A18	AF317653	G protein-coupled receptor 62
GPR63	A18	AF317654	G protein-coupled receptor 63
GPR72	A09	NM_016540	G protein-coupled receptor 72, Jp05
GPR73	A09	AAE24084	G protein-coupled receptor 73
GPR75	A09	NM_006794	G protein-coupled receptor 75
GPR80	A11	AF411109	G protein-coupled receptor 80
GPR81	A11	AF411110	G protein-coupled receptor 81
GPR85	A18	NM_018970	G protein-coupled receptor 85, Sreb2
GPR86	A12	NP_076403	Adp receptor
GPR87	A12	NM_023915	G protein-coupled receptor 87
GPR88	A18	NM_022049	G protein-coupled receptor 88
GPR91	A11	NM_033050	G protein-coupled receptor 91
GPR101	A18	NM_054021	G protein-coupled receptor 101
GPR102	A17	NM_053278	G protein-coupled receptor 102
GPR103	A06	AF411117	G protein-coupled receptor 103
GPRC	A13	P47775	Gpr12
GPRF	A03	P49685	Gpr15, Bob
GPRJ	A09	Q15760	Gpr19, Gpr-Nga
GPRL	A18	Q99679	Gpr21
GPRM	A06	Q99680	Gpr22
GPRV	A11	O00270	Gpr31
GPRW	A08	O75388	Gpr32
HM74	A11	P49019	G protein-coupled receptor Hm74
KI01	A12	Q15391	Udp-Glucose receptor, Kiaa0001
LSHR	A10	P22888	Lutropin-choriogonadotropic hormone receptor, Lh/Cg-R, Lsh-R, luteinizing hormone receptor, Lhcgr, Lhrhr, Lcgr
MAS	A08	P04201	Mas proto-oncogene, Mas1
ML1A	A09	P48039	Melatonin receptor Type 1a, Mel-1a-R, Mtnr1a
ML1B	A09	P49286	Melatonin receptor Type 1b, Mel-1b-R, Mtnr1b
ML1X	A09	Q13585	Melatonin-related receptor, H9, Gpr50
MRG	A08	P35410	Mas-related G protein-coupled receptor
NMU1R	A07	AF272362	Neuromedin U receptor 1, Nmur1, Gpr66, Fm-3
NTR1	A07	P30989	Neurotensin receptor Type 1, Nt-R-1, Ntsr1, Ntrr
NTR2	A07	O95665	Neurotensin receptor Type 2, Nt-R-2, levocabastine-sensitive neurotensin receptor, Ntr2 receptor, Ntsr2
NY1R	A09	P25929	Neuropeptide Y receptor Type 1, Npy1-R, Npy1r, Npyr, Npyy1
NY2R	A09	P49146	Neuropeptide Y receptor Type 2, Npy2-R, Npy2r
NY4R	A09	P50391	Neuropeptide Y receptor Type 4, Npy4-R, Pancreatic Polypeptide receptor 1, Pp1, Ppyr1,
Npy4r
P2Y5	A15	P43657	P2y purinoceptor 5, P2y5, purinergic receptor 5, P2ry5, 6h1
P2Y7	A05	Q15722	P2y purinoceptor 7, P2y7, Leukotriene B4 receptor, Chemoattractant receptor-like 1, P2ry7, P2y7, Gpr16, Cmkrl1, Ltb4r
P2Y9	A15	Q99677	P2y purinoceptor 9, P2y9, purinergic receptor 9, Gpr23, P2ry9
P2Y10	A15	AF000545	Putative purinergic receptor P2y10
P2Y12	A12	AF313449	Adp receptor, Sp1999
PAFR	A12	P25105	Platelet Activating Factor receptor, Paf-R, Ptafr
PNR	A17	AF021818	Putative neurotransmitter receptor
PSP24	A18	U92642	High-affinity lysophosphatidic acid receptor homolog, Gpr45
RDC1	A02	P25106	G protein-coupled receptor Rdc1 homolog
RE2	A18	AF091890	G protein-coupled receptor Re2
SALPR	A05	NM_016568	Somatostatin and angiotensin-like peptide receptor, Loc51289
SREB3	A18	NM_018969	Super conserved receptor expressed in brain 3
TM7SF1	A01	AF027826	Putative seven pass transmembrane protein
TSHR	A10	P16473	Thyroid stimulating hormone receptor, thyrotropin receptor, Tsh-R
Human GPCR - Family B
EMR1	B	Q14246	Cell surface glycoprotein emr1, Emr1 hormone receptor
EMR2	B	AF114491	Egf-like module Emr2
EMR3	B	AF239764	Egf-like module-containing mucin-like receptor Emr3
BAI1	B	O14514	Brain-specific angiogenesis inhibitor 1
BAI2	B	O60241	Brain-specific angiogenesis inhibitor 2
BAI3	B	O60242	Brain-specific angiogenesis inhibitor 3, Kiaa0550
GPR56	B	NM_005682	G protein-coupled receptor 56
Human GPCR - Family C
GPRC5B	C	NM_016235	G PROTEIN-COUPLED RECEPTOR, FAMILY C, GROUP 5, MEMBER B, GPRC5B
GPRC5C	C	NM_018653	G protein-coupled receptor, family C, group 5, member C, Gprc5c
GPRC5D	C	NM_018654	G protein-coupled receptor, family C, group 5, member D, Gprc5d

A complete list is supplied as additional data file. Orphan receptors are shown in bold.

Figure 1

An example multiple sequence alignment of seven receptors. Protein sequences of GPR87, KI01, GPR86, P2Y12, H963, GPR34 and PAFR belonging to subgroup 12 were aligned with ClustalX and modified by deleting the extremely variable amino termini upstream of the first transmembrane domain and carboxyl termini downstream of the seventh transmembrane domain as indicated. Identical amino-acid residues in all aligned sequences are shaded in black and similar residues in gray. Transmembrane (TM) domains identified by the TMpred program are indicated.

Phylogenetic analysis

Because of the large number of sequences in family A, we had to use a combination of computational methods to accomplish the best possible description of their phylogenetic relationship. In a first step we used the distance-based neighbor-joining method as the only one computationally feasible. Neighbor joining has been shown to be efficient at recovering the correct tree topology [21], but is greatly influenced by methodological errors, for example, the sampling error [22]. This can in part be overcome by bootstrapping, a method of testing the reliability of a dataset by the creation of pseudoreplicate datasets by resampling. Bootstrapping assesses whether stochastic effects have influenced the distribution of amino acids [23]. In previous publications on this topic, bootstrapping has not been generally used.

We generated a neighbor-joining tree of family-A sequences, and considered tree branches to be confirmed if they were found in more than 500 of 1,000 bootstrap steps (Figure 2). The same branching pattern was found by least squares (data not shown) as implemented in FITCH [24], but it was not possible to compute enough bootstrap steps with the equipment used. The remaining sequences of unconfirmed branches were then assigned to existing branches according to results obtained with the local alignment tool BLASTP (see Additional data files) [25] to account for similarities in parts of the sequences not sufficient for repeated global alignment. The p-value was used as a measure of similarity.

Figure 2

Neighbor-joining tree of the rhodopsin receptor-like family A inferred from the multiple sequence alignment using PHYLIP 3.6. Support values for each internal branch were obtained by 1,000 bootstrap steps, and are indicated. Pairwise distances were determined with PROTDIST and the JTT substitution frequency matrix. The tree was calculated with NEIGHBOR using standard parameters and rooted with the distant, though related, family-B receptor GPRC5B as the outgroup. The consensus tree of all bootstrapped sequences was obtained with CONSENSE. Orphan receptors are shown in bold. Scale bar indicates the branch length of 100 substitutions per site.

As this strategy still left four subgroups too large for detailed analyses, we recalculated neighbor-joining trees and in some cases least-square trees of these sequences to create subgroups A1 and 2, A4 and 5, A11 and 15 and A17 and 18. This approach finally resulted in 19 differently sized subgroups of family A (Table 2) that were further subjected to the more reliable maximum-likelihood and quartet-puzzling algorithms. Maximum-likelihood approaches calculate the probability of the observed data assuming that it has evolved in accordance with a chosen evolutionary model. Phylogenies are then inferred by finding trees and parameters that yield the highest likelihood. Maximum-likelihood approaches tend to outperform alternative methods such as parsimony or distance-based methods. The main advantage is the application of a well defined model of sequence evolution to a given dataset [26]. Maximum likelihood is the estimation method least affected by sampling error and tends to be robust to many violations of the assumptions in the evolutionary model. The methods are statistically well founded, evaluate different tree topologies and use all sequence information available [27,28]. Because of their smaller size, families B and C could be subjected to these methods without prior subgrouping. This resulted in 19 phylogenetic trees, comprising 241 receptors for family A (Figures 3,4,5,6), one tree from 23 sequences for family B and one tree from 14 sequences for family C (Figure 7). Family-A trees were rooted with the human family-B receptor GPRC5B and families B and C with family-A receptor 5H1A. The sequence used to root the tree (the outgroup) is supposed to be a distant, though related, sequence. In some of our groups, the phylogenetic trees could not be fully resolved. This could be due to either very similar or very distant sequences. In both cases the phylogenetic signal is too weak to resolve the tree [29]. Several receptors (for example, TM7SF1, DUFF, GPR, GPRM, GPR75, GPR88, MAS and MRG) were found to be only distantly related to other known receptors used in our analysis. A possible explanation could be the previously proposed convergent evolution of this large protein family, meaning that these receptors have acquired the compelling similarity in their overall structures as a result of functional need, not phylogenetic relationship. The lack of significant sequence similarity among the different GPCR families favors this assumption [30,31,32]. Other explanations for the lack of significant sequence similarities might be an extraordinary divergence (genetic drift) or technical problems of the sequence-analysis methods used in analyzing polytopic membrane proteins or large protein families [33].

Table 2

Receptor subgroups derived from a combination of neighbor-joining and BLASTP results

A1	A2	A3	A4	A5	A6	A7	A8	A9	A10	A11
C3X1	ADMR	AG22	GPR7	GALR	FF1R	BRS3	C3AR	GPR72	FSHR	GPR40
CKR1	BONZO	AG2R	GPR8	GALS	FF2R	ET1R	C5AR	GPR73	GPR48	GPR41
CKR2	CCR11	AG2S	OPRD	GALT	GASR	ETBR	C5L2	(GPR75)	GPR49	GPR42
CKR3	CCR3	APJ	OPRK	GPR54	(GPR)	ETBR-LP2	CML1	GPRA	LSHR	GPR43
CKR4	CCR4	BRB1	OPRM	GPRO	GPR103	GHSR	FML1	GPRJ	TSHR	GPR80
CKR5	CCR5	BRB2	OPRX	P2Y7	(GPRM)	GPR37	FML2	ML1A		GPR81
CKR8	CKR6	GPR25	SSR1	SALPR	GRHR	GPR38	FMLR	ML1B		GPR82
CKRX	CKR7	GPRF	SSR2	UR2R	OX1R	GPR39	GPR1	ML1X		GPR91
CXC1	CKR9		SSR3		OX2R	GRPR	GPR44	NK1R		GPRV
(TM7SF1)	CKRA		SSR4		OXYR	NMBR	GPRW	NK2R		HM74
	CML2		SSR5		V1AR	NMU1R	(MAS)	NK3R		P2UR
	(DUFF)				V1BR	NMU2R	(MRG)	NK4R		P2Y11
	IL8A				V2R	NTR1		NY1R		P2Y4
	IL8B					NTR2		NY2R		P2Y6
	RDC1					TRFR		NY4R		P2YR
								NY5R
A12	A13	A14	A15	A16	A17	A18	A19		B	C
GPR34	ACTR	PD2R	EBI2	OPSB	5H2A	AA1R	5H1A		BAI1	CASR
GPR86	CB1R	PE21	G2A	OPSD	5H2B	AA2A	5H1B		BAI2	GBR1
GPR87	CB2R	PE22	GPR35	OPSG	5H2C	AA2B	5H1D		BAI3	GBR2
H963	EDG1	PE23	GPR4	OPSR	5H6	AA3R	5H1E		CALR	GPRC5B
KI01	EDG2	PE24	GPR55	OPSX	A1AA	ACM1	5H1F		CD97	GPRC5C
P2Y12	EDG3	PF2R	GPR65	RGR	A1AB	ACM2	5H5A		CGRR	GPRC5D
PAFR	EDG4	PI2R	GPR68		A1AD	ACM3	5H7		CRF1	MGR1
	EDG5	TA2R	GPR92		A2AA	ACM4			CRF2	MGR2
	EDG6		GPRH		A2AB	ACM5			EMR1	MGR3
	EDG7		GPRI		A2AC	GPR101			EMR2	MGR4
	EDG8		GPRK		A2AD	GPR27			EMR3	MGR5
	GPR3		P2Y10		B1AR	GPR52			GIPR	MGR6
	GPR6		P2Y5		B2AR	GPR61			GLPR	MGR7
	GPRC		P2Y9		B3AR	GPR62			GLR	MGR8
	MC3R		PAR2		D2DR	GPR63			GPL2
	MC4R		PAR3		D3DR	GPR78			GPR56
	MC5R		THRR		D4DR	GPR84			GRFR
	MSHR				DADR	GPR85			PACR
					DBDR	(GPR88)			PTR2
					GPR102	GPRL			PTRR
					GPR57	HH1R			SCRC
					GPR58	PSP24			VIPR
					HH2R	RE2			VIPS
					PNR	SREB3

Very distantly related receptors that are possibly not phylogenetically related are shown in brackets. Orphan receptors are shown in bold.

Figure 3

Chemokine receptors (subgroups A1 and A2). Phylogenetic trees of the subgroups were inferred using Puzzle 5.0 corrected by the JTT substitution frequency matrix. Quartet-puzzling support percentage values from 10,000 puzzling steps are shown. The scale bars indicate a maximum likelihood branch length of 0.1 inferred substitutions per site. Orphan receptors are shaded.

Figure 4

Peptide receptors (subgroups A3-A10). The scale bar indicates a maximum likelihood branch length of 0.1 inferred substitutions per site. Orphan receptors are shaded. For method see Figure 2

Figure 5

Nucleotide and lipid receptors (subgroups A11-A16). The scale bar indicates a maximum-likelihood branch length of 0.1 inferred substitutions per site. Orphan receptors are shaded. For method see Figure 2.

Figure 6

Biogenic amine receptors (subgroups A17-A19). The scale bar indicates a maximum-likelihood branch length of 0.1 inferred substitutions per site. Orphan receptors are shaded. For method see Figure 2.

Figure 7

Families B and C of the G-protein-coupled receptors (GPRCs). Phylogenetic trees of families B and C were inferred using Puzzle 5.0 corrected by the JTT substitution frequency matrix. Quartet-puzzling support percentage values from 10,000 puzzling steps are shown. The scale bar indicates a maximum likelihood branch length of 0.1 inferred substitutions per site. Orphan receptors are shaded.

Receptor family A subgroups

In contrast to the subfamilies presented in GPCRDB [34], a database widely used in the field, our grouping shows the orphan receptors within their respective subgroup and their relationship to receptors with known ligands. In addition, our method sometimes resulted in subgroups with members whose ligands belong to different substance classes. These results are discussed in more detail below.

Chemokine receptors

Groups A1 and A2 comprise the chemokine receptors (Figure 3). The chemokine ligand superfamily is defined by four conserved cysteines that form two disulfide bonds, and can be structurally subdivided into two major branches based on the spacing of the first cysteine pair. Chemokines in which these residues are adjacent form the CC subfamily (corresponding to the SWISS-PROT CKR nomenclature used here), and those separated by a single amino acid comprise the CXC subfamily (here CCR and IL8R; for a review see [35]). We had to divide the whole subfamily into two groups to perform a detailed phylogenetic analysis. This sub-grouping produced the same dichotomy, as suggested by the two-ligand motifs, as another example of the parallel evolution of receptors and ligands. Similar results describing this parallel evolution were found previously using a different computational approach [36].

Group A1 mainly comprises the CC family. We hypothesize that the orphan receptor CKRX, which constitutes a separate branch related to CKR1, 2, 3 and 5, might also bind a CC ligand. In contrast, TM7SF1 in this group seems to be only distantly, if at all, related to family-A receptors. It was grouped according to BLASTP results, where a misleading local alignment of approximately 20 amino acids placed it in the vicinity of the chemokine receptors. Group A2 is more heterogeneous and comprises receptors for CC and CXC ligands, as well as an orphan receptor (ADMR) previously thought to bind the peptide adrenomedullin. Adrenomedullin has now been shown to bind a family-B receptor and is discussed further below. The orphan receptor RDC1 in group A2 was first believed to be a receptor for vasointestinal peptide VIP [37], a notion not supported by phylogeny and later dismissed by experimental data [38]. Our results place it closer to the ADMR receptor than to the typical chemokine receptors. CML2 is a typical, but distant, member of the chemokine receptor family. The DUFF receptor (the Duffy antigen) is also very distantly related and was only grouped into A2 by BLASTP results.

Peptide receptors

Group A3 consists of receptors for the small peptides angiotensin (8 amino acids), bradykinin (9 amino acids) and apelin (Figure 4). Four forms of apelin (12, 13, 17 and 36 amino acids) have been described, but only those of 12 and 13 amino acids bind in nanomolar concentrations [39]. The orphan receptors GPRF and GPR25 in this group are related as closely to the apelin receptor APJ as to the angiotensin or bradykinin receptors, and might also bind small peptides. GPRF acts as a co-receptor for the human immunodeficiency virus (HIV) [40], like the APJ receptor [41], which further hints at structural homology of the two ligands. Opioid and somatostatin receptors make up group A4. Both somatostatin and opioid peptides are derived from the processing of larger precursors. The somatostatins are cyclic peptides of 14 and 28 amino acids. The opioid precursors preproenkephalin, preprodynorphin, prepro-opiomelanocortin and prepronociceptin display a strikingly similar general organization and a conserved amino-terminal region that contains six cysteines, probably involved in disulfide bond formation.

The processed neuropeptides, in contrast, are less similar to each other. It could be speculated that the receptors first bound the precursors themselves, and that the diversity derived from processing is evolutionarily new. Processing prepronociceptin gives rise to two evolutionarily conserved peptides besides orphanin FQ, the ligand for OPRX. It has not been reported whether these peptides bind to the orphan receptors GPR7 and GPR8, which constitute a new branch related to the opioid receptors.

In group A5 we find three receptors that bind the 30-amino-acid peptide galanin, and related to these the GPR54 receptor, which is activated by the 54-, 14-, and 13-amino-acid peptides derived from the product of KiSS-1, a metastasis suppressor gene for melanoma cells. These kisspeptins all share a common RF-amide caboxyl terminus. Although only distantly related to each other, both GPRO (melanin-concentrating hormone) and UR2R (urotensin II peptide) bind cyclic peptides originally isolated from fish. Similarly distant is the orphan receptor SALPR, which shares sequence similarity with somatostatin (A4) and angiotensin (A3) receptors, but subgrouping of groups A4 and 5 by neighbor joining led to its placement in group 5. SALPR does not bind somatostatin or angiotensin ligands [42], but could bind another cyclic peptide. The P2Y7 receptor in group A5 does not bind nucleotides [43], as suggested by the name, but was published as a receptor for the lipid leukotriene 84 [44], a notion not supported by phylogeny. In addition, two new leukotriene receptors - CLT1 and CLT2 - have been cloned and characterized during the preparation of this manuscript [45,46] and were found to be unrelated to P2Y7.

Group A6 is again composed solely of receptors for peptide ligands. The orphan receptor GPR103 is related to the neuropeptide FF receptors that bind two amidated mammalian neuropeptides - NPAF (A-18-F-amide) and NPFF (F-8-F-amide), also known as morphine-modulating peptides. These peptides, which may also be the ligand for GPR103, are members of a large family of neuropeptides related to the molluscan cardioexcitatory neuropeptide (FMRF-amide, Phe-Met-Arg-Phe-amide). The orphan receptors GPRM and GPR in group A6 are most probably also peptide receptors, but are only very distantly related to the others and show no relationship to receptors with known ligands. Group A7 is also composed of receptors for peptide ligands: neuromedin, neurotensin, motilin, endothelin, bombesin and the releasing hormones for growth hormone and thyrotropin. GPR39 might bind a small peptide ligand like the closely related neurotensin receptors NTR1 and 2, which binds a 13-amino-acid peptide derived from a larger precursor protein. GPR37 and ETBR-LP2 are related to each other and branch off the endothelin receptors that bind characteristic bicyclic peptides of 21 amino acids containing four cysteines linked by two disulfide bonds.

Group A8 has two branches with receptors with known ligands. These receptors bind the structurally diverse but functionally related chemotactic substances N-formylmethionyl and the anaphylatoxic complement factors. The N-formylmethionyl ligands are small hydrophilic peptides of bacterial origin, but recently a number of new peptide agonists have been identified that selectively activate the high-affinity fMLF receptor FPR and/or its low-affinity variant FPRL1. These agonists include peptide domains derived from the envelope proteins of HIV type 1 and at least three amyloidogenic polypeptides, the human acute-phase protein serum amyloid A, the 42-amino-acid form of beta-amyloid peptide and a 21-amino-acid fragment of the human prion protein. Furthermore, a cleavage fragment of neutrophil granule-derived bactericidal cathelicidin, LL-37, is also a chemotactic agonist for FPRL1 (for a review see [47]). The complement factors C3a and C5a are large but highly hydrophilic proteins with a mainly alpha-helical structure held together by three disulfide bridges. C5a is rapidly desarginated to the less potent derivative C5adR74, which is the ligand for the C5L2 receptor. The orphan receptors GPR1, CML1 and GPR44 all cluster, and constitute a separate branch as distant as the other two branches. No prediction of the possible structure of the ligands for these receptors can be derived from this tree, but maybe they will function as chemotactic peptides. This could at least hint at leukocytes or inflamed tissue as a possible source for these ligands. The receptor GPRW constitutes its own branch, not as distant to the main group as the MAS oncogene product and the related receptor MRG, which are only very distantly related to the group.

All receptors in group A9 with known ligands bind peptides, except for a side branch consisting of receptors for the biogenic amine melatonin. The orphan receptor ML1X is closely related to melatonin receptors ML1A and B, but apparently does not bind melatonin [48]. GPR73 is related to the neuropeptide Y (NPY) receptor NY2R which mainly binds the pancreatic peptide YY of 36 amino acids, and these two are placed together on a branch distinct from the NPY receptors NY4R and NY1R. GPR73 does not bind the NPY ligand family [49], but possibly a similar large peptide ligand. The orphan receptors GPR72 and GPRJ constitute a new subgroup that most probably bind related peptide ligands. GPR72 does not bind a NPY ligand [49]. GPR75 is only very distantly related to the whole A9 group. The receptors for the glycoprotein hormones thyroid-stimulating hormone (TSH), luteinizing hormone (LSH) and follicle-stimulating hormone (FSH) make up Group A10. GPR48 and 49 are very similar in their overall structure, with long amino termini, but their relationship is also evident in the neighbor-joining tree constructed from alignments without amino and carboxyl termini. It has been recently shown that these receptors mediate the action of relaxin, a peptide hormone of the insulin-like growth factor family secreted by the corpus luteum during pregnancy [50].

Nucleotide and lipid receptors

The receptors with known ligands in group A11 are the P2Y receptors, which bind pyrimidine as well as purine nucleotides (Figure 5). Several orphan receptors constitute new clusters. GPR80 and GPR91 are distantly related to each other and relatively close to the P2Y receptors. GPR80 is the closest relative of the newly identified CLT2 receptor for leukotrienes as judged by BLASTP results. GPR81, HM74 and GPRV and GPR 40-43 belong to branches only distantly related to P2Y receptors. Within these potential new subfamilies, GPR41-43, GPR81 and HM74 are more closely related to each other than to GPR40 (for GPR41-43) and GPRV (for GPR81 and HM74).

In group A12, the platelet-activated receptor, a lipid receptor and receptors activated by nucleotides mingle, but are found on different side branches. The orphan receptor GPR87 is closely related to the receptor for UDP-glucose KI01 and to the ADP-binding receptors P2Y12 and GPR86. We assume that this receptor might also bind UDP-glucose or another modified nucleotide. GPR34 is distantly related to the platelet-activating factor (PAF) receptor; it was not activated by available lipid ligands [51], but might nevertheless bind a lipid ligand. Group A13 contains both peptide and lipid receptors but they make up different branches. The peptide branch binds peptides derived from the processing of pro-opiomelanocortin that gives rise to peptides of between 12 and 36 amino acids. The EDG and cannabinoid receptors constitute clusters, and one cluster distinct from the other three consists of the orphan receptors GPR3, GPR6 and GPRC, which have been grouped closer to the lipid EDG receptors in the overall neighbor-joining tree (Figure 2). This information helped to identify a phospholipid ligand for GPRC (H. Chica Schaller, personal communication).

The receptors in group A14 all bind ligands derived from arachidonic acid by the action of cyclooxygenase. These receptors for lipid-derived autacoids or prostanoids comprise receptors for the prostaglandins and thromboxanes. There are no orphan receptors in this group. Group A15 is a very heterogenous group composed of receptors for the lipids sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC) and psychosine, and receptors activated by proteases. GPR4 and GPR68 both bind SPC, like the EDG receptor branch consisting of the EDG1, 3, 6 and 8 receptors in A13, but are not closely related. Protease-activated receptors become activated by a part of the former amino terminus cleaved by the protease. The new amino terminus then functions as a tethered ligand and activates the receptor. This can be mimicked by very small peptides derived from this ligand; such receptors should therefore rather resemble peptide receptors. The orphans P2Y5, P2Y9 and P2Y10 receptors were not placed in group 11 and 12 like most P2Y receptors, but in group A15, supporting the fact that they were misnamed. P2Y5 and P2Y9 do not bind nucleotides [52,53], but this has not been shown yet for P2Y10. All other orphan receptors in this group, with the exception of GPR35 and GPR55 which cluster together, are as distantly related to each other as to the receptors with known ligands. Group A16 contains the opsins, receptors that are activated by isoprenoid ligands, and no orphan receptors.

Biogenic amine receptors

Some serotonin receptors and receptors for the biogenic amines adrenaline, dopamine and histamine are all placed on different branches in group A17 (Figure 6). An additional branch consists of the orphan receptors GPR102, PNR, GPR57 and GPR58, which are as distantly related to the others as, for example, is the alpha-adrenergic receptor branch. PNR and GPR58 expressed in COS cells did not bind various serotonin receptor-specific ligands [54]. Their ligands might be small molecules with similar properties. Group A18 is very heterogeneous and consists of receptors for the biogenic amines acetylcholine and adenosine, and the HH1R receptor for histamine, as well as many distantly related orphan GPCRs. GPR63 is closely related to the orphan receptor PSP24. The Xenopus laevis homolog of this receptor binds LPA [55]. GPR101 and RE2, GPRL and GPR52, and GPR61 and GPR62 constitute their own subgroups. In particular, the SREB1-3 cluster (GPR85, GPR27 and SREB3) makes up its own family, with only a distant relationship to other GPCRs in this group. No orphan receptors are found in group A19, which consists entirely of serotonin receptors distinct from those in A17.

During the preparation of this manuscript several new family-A receptors that could not be fitted into our analysis were identified. These comprise 15 new receptors distinct from the classical biogenic amine receptors that apparently bind the trace amines tyramine, β-phenylethylamine, tryptamine and octopamine [56]. In addition, a new subfamily of GPCRs related to the mas oncogene and uniquely expressed in small nociceptive sensory neurons were shown to be the receptors for a number of enkephalin fragments [57].

Receptor families B and C

Family B (Figure 7) was named after the secretin receptor. Yet proteins showing homology to this receptor make up only one of four distantly related subgroups. The receptors EMR1, EMR2 and EMR3, and the CD97 surface antigen, all have several epidermal growth factor (EGF)-like domains in the extracellular amino terminus. They constitute their own cluster only distantly related to the rest of the family. The same applies to the brain-specific angiogenesis inhibitor family BAI1-3. GPR56 was assigned to family B because it shows the typical signature [58], but is so far the only one of its kind. So far no non-protein ligand has been identified as a ligand for family-B receptors. Astonishingly, one family-B receptor, namely the CGRP receptor, requires coexpression with single transmembrane receptor activity-modifying proteins (RAMP1-3) for ligand binding and signal transduction [59]. Coexpression of different RAMPs results in binding of different cyclic peptide ligands such as adrenomedullin, amylin or the calcitonin gene-related peptide (for a review see [60]). This could further complicate the identification of the cognate ligands for these family-B orphan receptors, but we assume that they will also bind large peptide ligands. In family C (Figure 7), the metabotropic glutamate receptors MGR1-8 bind the small molecule glutamate, the CASR receptor senses extracellular calcium concentration, and receptors GBR1-2 bind the small molecule gamma-amino butyric acid (GABA). GPRC5B, C and D constitute their own subgroup with no closer relationship to the other members, but might also bind small molecules.

Conclusions

In this work, we calculated the phylogenetic distances of 277 human GPCRs and show the relationship of orphan receptors to receptors for known ligands with support values for each branch. We then grouped orphan receptors and receptors with known ligands into 19 subgroups that sometimes differ from previous classifications. Three subgroups are composed of receptors for ligands that belong to different substance classes; for example, in group A12, lipid receptors and receptors activated by nucleotides mingle, and in groups A13 and A15, peptide and lipid receptors. In both subgroups the receptors binding ligands of different substance classes make up different branches. We hope that this approach proves valuable for identifying the natural ligands of orphan receptors, as related receptors have previously been shown to have ligands with similar structural features.

Materials and methods

Sequence database mining

A database search excluding olfactory and gustatory receptors identified the amino-acid sequences of 281 human GPCRs. Only sequences annotated as GPCRs in the following databases were used: NCBI [61], SWISS-PROT [62], EMBL [63] and GPCRDB [34,64]. Receptors without published ligands in PubMed [65] were defined as orphan GPCRs.

Multiple sequence alignments

Multiple protein sequences were aligned with ClustalX 1.81 [66]. Pairwise alignment parameters were set as: slow/accurate alignment; gap opening penalty 10; gap extension penalty 0.10; protein weight matrix BLOSUM 30. Multiple alignment parameters were set as: gap opening penalty 10; gap extension penalty 0.05; delay divergent sequences 35%; protein weight matrix BLOSUM series [67]. The alignments were modified by deleting the extremely variable amino termini upstream of the first transmembrane domain and carboxyl termini downstream of the seventh transmembrane domain. Alignment editing and shading was done using BioEdit Sequence Alignment Editor [68] and GeneDoc Multiple Sequence Alignment Editor [69]. Transmembrane domains were identified using the TMpred program [70] and, where available, data from the original publication [71].

Clustering of subgroups

An overall phylogenetic tree of family A was inferred from the multiple sequence alignment with PHYLIP 3.6 [72]. Bootstrapping was performed 1,000 times using SEQBOOT to obtain support values for each internal branch. Pairwise distances were determined with PROTDIST and the JTT substitution frequency matrix [73]. Neighbor-joining phylogenetic trees [21] were calculated with NEIGHBOR using standard parameters. The human GPRC5B receptor belonging to family B was used as outgroup for family A. The out-group sequence is supposed to be a distant, though related, sequence and is used to root the tree. The majority-rule consensus trees of all bootstrapped sequences were obtained with the program CONSENSE. Representations of the calculated trees were constructed with TreeView [74]. Clusters with bootstrap values greater than 50% were defined as confirmed subgroups, and sequences with lower values added to these subgroups according to their sequence similarity in the alignment as judged by visual inspection and the results of pairwise local alignments with all other sequences by BLASTP [25]. The p-value was used as a measure of similarity.

Quartet-puzzling trees

Multiple protein sequence alignments of these new subgroups were created as described above. Phylogenetic trees were inferred from these alignments using Puzzle 5.0 [75] to calculate maximum-likelihood distances corrected by the JTT substitution-frequency matrix [73] with amino-acid usage estimated from the data, site-to-site rate variation modeled on a gamma distribution with eight rate categories plus invariant sites, and the shape parameter estimated from the data. The human GPRC5B receptor of family B was used as an outgroup for family A. The human 5H1A receptor of family A was used as an outgroup for families B and C (the outgroups are not shown in the figures here). Quartet-puzzling (QP) trees were constructed with the described settings and 10,000 puzzling steps to obtain support values (QP reliability) for each internal branch. The program Puzzle 5.0 was used in a parallelized version (ppuzzle) with a message-passing interface (MPI) implementation on a HP 9000 N-Class Enterprise Server Cluster consisting of five HP 9000 N-Class shared-memory multiprocessor systems with eight PA-RISC 8600 (552 MHz) processors each. Representations of the quartet-puzzling trees were constructed with TreeView [74].

Additional data files

Additional data files available with this paper include a data table with names, synonyms and accession numbers of all GPCRs, and the BLASTP results of all GPCRs (full-length sequences and sequences without amino or carboxyl termini).

Additional data file 1

A data table with names, synonyms and accession numbers of all GPCRs

Click here for additional data file

Additional data file 2

Names, synonyms and accession numbers of all GPCRs, and the BLASTP results of all GPCRs full-length sequences

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Additional data file 3

Names, synonyms and accession numbers of all GPCRs, and the BLASTP results of all GPCRs sequences without amino or carboxyl termini

Click here for additional data file