Chemokine receptors

Groups A1 and A2 comprise the chemokine receptors (Figure ​(Figure3).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 ​(Figure4).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 ​(Figure5).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 ​(Figure2).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.