Use of Sequence Homology Cross Genome Phylogenetic Analysis and Chemogenomics to Predict Candidate Ligands

Sequence comparison between orphan 7TM proteins and GPCRs with known ligands has always been a straightforward strategy for candidate ligand prediction. Based on simple sequence comparisons, several orphan 7TM proteins

TABLE 7.1 Deorphanized 7TM Proteins since 2005

GPCRs

Ligands

Assay

Deorphanization Strategy

Reference

GPR1

Chemerin

Enzyme Fragment Complementation

P-arrestin recruitment

[70]

GPR17

Uracil nucleotides/

[35S]GTPyS

Phylogenetic analysis

[28]

cy cte iny 1-le uko trie ne s

GPR18

TV-arachidonylglycine

Ca2+ (Fluo-3)

Reverse pharmacology

[107]

GPR34

Lysophophatidylserine

cAMP (EIA kit), [35S]GTPyS

Phylogenetic analysis

[108]

GPR35

Kynurenic acid

Ca2+ (aequorin)

Reverse pharmacology

[17]

GPR55

Canabinoid ligands

Ligand binding, [35S]GTPyS

Phylogenetic analysis

[109]

GPR75

RANTES

cAMP (luciferase reporter)

Reverse pharmacology

[19]

GPR77

Acylation-stimulating protein

P-arrestin translocation

Fluorescence-based

[65]

P-arrestin translocation

assay

GPR84

Medium-chain free fatty acids

Ca2+ (aequorin)

Reverse pharmacology

[18]

GPR87

Lysophosphatidic acid

GPR87-Gai6 fusion protein, Ca2+

Reverse pharmacology

[56]

(Fura-2)

GPR92

Lysophophatidic acid

Ligand binding, cAMP (EIA kit)

Phylogenetic analysis

[110]

Ligand binding, internalization (IF)

Phylogenetic analysis

[111]

GPR119

Lysophophatildylcholine

cAMP (HTRF kit)

Reverse pharmacology

[112]

GPR120

Free fatty acids

GPR120-Gal6 fusion protein.

Reverse pharmacology

[57]

internalization (flow cytometry

and IF)

RDC1 (CXCR7)

CXCL12

Ligand binding, internalization (IF)

Phylogenetic analysis

[113]

GPRC6A

L-alpha-amino acids (L-Arg,

X-oocytes, CA2+ (Fluo-4)

Chemogenomics

[30]

L-Lys, L-omithine)

P2Y5 receptor

Lysophosphatidic acid

cAMP (luciferase reporter)

Reverse pharmacology

[114]

P2Y10 receptor

Sphingosine -1 -phosphate

Ca2+ (Fura-2)

Phylogenetic analysis

[27]

Lysophosphatidic acid

IE, Immunofluorescence; EIA, enzyme-linked immunoassay, HTRF, homogenous time-resolved fluorscence; X-oocytes, Xenopus oocytes.

IE, Immunofluorescence; EIA, enzyme-linked immunoassay, HTRF, homogenous time-resolved fluorscence; X-oocytes, Xenopus oocytes.

have been classified in the past into GPCR subfamilies and subsequently deorphanized [6, 24]. The complete sequencing of the human genome and the emergence of new bioinformatic tools further accelerated and refined this process. According to the most recent classification, some 7TM proteins segregate into subfamilies exclusively composed of orphans, and others into subfamilies with known ligands [5, 3]. Sequencing of the genome of many different species stimulated cross genome phylogenetic analysis that provided further insights in the correct classification of orphan 7TM proteins [25, 26].

Recent examples where phylogenetic analysis had a major impact on the deorphanization process are two P2Y-related receptors, the P2Y10 receptor and GPR17 [27, 28]. GPR17 constitutes an interesting example as this protein occupies an intermediate position between purinergic P2Y and cysteinyl-leukotrienes (CysLTs) receptors. Functional studies showed that GPR17 indeed seems to straddle both subfamilies and binds to both uracil nucleotides and CysLTs with high affinity (Table 7.1).

Further information came from a chemogenomic analysis of the ligand binding pocket of human non-odorant GPCRs [29]. Interestingly, clustering of 30 residues predicted or known to be important for ligand binding was similar but not identical compared to the phylogenetic tree derived from full-length GPCR cDNAs. This approach was used to deorphanize GPRC6A as the receptor for L-a-amino acids [30] and will certainly help to predict potential ligands for other 7TM proteins in the future (Fig. 7.1).

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