Controversial Issues

Deorphanization of 7TM proteins has become increasingly difficult. New screening strategies will help to deorphanize further orphans, and new concepts may highlight additional functions that may or may not depend on ligand binding to the predicted orthosteric binding site. However, deorphanization of the remaining orphan GPCRs is expected to remain a slow and tedious task.

From the early days of deorphanization on, researchers have been often confronted with high levels of apparent constitutive activity when expressing cDNAs of orphan 7TM proteins. Although many GPCRs exhibit a significant level of constitutive activity, it should be mentioned that in the case of orphan 7TM proteins, the possibility always remains that the apparent constitutive activity might actually reflect the presence of an endogenous ligand that either is difficult to remove or which is produced by the cell. This is illustrated by the adenosine A2 receptor whose apparent constitutive activity could be explained by endogenous adenosine [96]. A recent study on GPR40 shows that its apparent constitutive activity is due to the occupation of the receptor binding site by endogenous fatty acid ligands [97]. Screening programs for inverse agonists should take into account such potential complications.

The deorphanization process is not always linear. Reports claiming the successful deorphanization have to be double- checked by other groups. Initial results are indeed not always confirmed, leading to a controversy, which may persist for several years before finding a consensus. At least three groups of 7TM proteins are currently at that state. The first case concerns the GPCR subfamily comprising GPR4,GPR68 (OGR1), GPR65 (TDAG8), and GPR132 (G2A), which have been initially deorphanized as receptors of lipid messengers. However, these data could not be confirmed by others and were retracted several years later. More recently, GPR4, GPR65, and GPR68 were reported to be proton-sensing receptors that activate either phosphoinositol or cAMP pathways [98]. The nature of the physiologically relevant ligand of the more distantly related GPR132 is still not clear. The second debated orphan 7TM protein is GPR39. In 2005, GPR39 was reported to be the receptor for obestatin, a 23 amino acid peptide derived from the ghrelin precursor protein [99]. However, 2 years later, several other groups could not confirm these findings [100, 101]. The last case, which is extensively debated in the literature, is GPR30. Controversial features include its subcellular localization, at the plasma membrane versus the endoplasmic reticulum, its capacity to bind to estrogens, and the discrimination between effects mediated though GPR30 and the classical nuclear steroid receptors. Despite numerous studies and the development of specific tools such as GPR30-selective ligands and antibodies directed against GPR30, no clear consensus has been reached today on all these issues, demonstrating the difficulties that may arise in the deorphaniza-tion process [102, 103].

Systematic screening for putative heterodimerization partners of orphan 7TM proteins may represent a tremendous effort as more than 100 orphans have to be matched with approximately 260 GPCRs with known ligand. Biotechnology companies such as Dimerix Bioscience have already set up screening procedures for the identification of GPCR heterodimers. Candidate heterodimers could then be further characterized in established functional assays (binding, trafficking, and signaling assays). In respect to the other non-GPCR proteins that associate with orphan 7TM proteins, a systematic screening of potential interaction partners is more difficult in view of the large diversity of these proteins.

Finally, the question whether at least some of the remaining orphans are "[rue orphans" that do not bind to any endogenous ligand was suggested recently [104], will be difficult to answer even in the presence of compelling evidence for ligand-independent function such as the modification of the func tion of other proteins by heterodimerization. However, in some cases, evolutionary analysis may provide meaningful insights. Evolutionary trace analysis of the putative GABA binding site of the orphan GABAB2 7TM protein indicated that this binding site is not under evolutionary pressure strongly supporting the absence of any ligand binding capacity [105]. Furthermore, evolutionary analysis of GPR50, which belongs to the melatonin receptor subfamily but does not bind to melatonin or any other known ligand, showed that GPR50 is the mammalian ortholog of Mellc, a high-affinity melatonin receptor in lower vertebrates [106] . This observation, together with the fact that the GPR50 gene evolved very rapidly in mammals, indicate that GPR50 has lost its capacity to bind melatonin during evolution. Although this finding does not completely rule out the possibility that GPR50 binds to another endogenous ligand, it strongly supports the idea that GPR50 is a true orphan receptor, whose expression during evolution is maintained due to essential ligand -independent functions.

In conclusion, new assays and concepts will certainly help to deorphanize many of the remaining orphan 7TM proteins; others might be true orphans with ligand-independent functions. Both categories, true orphans and deorphanized GPCRs, are likely to have great potential for drug development.

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