Cellular and Molecular Biology of Orphan G Protein‐Coupled Receptors

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The superfamily of G protein‐coupled receptors (GPCRs) is the largest and most diverse group of membrane‐spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of ∼168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so‐called orphan GPCRs. Eighty‐six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR‐mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

Introduction

G protein‐coupled receptors (GPCRs) are the largest family of cell surface molecules. They allow tissues to respond to a wide variety of extracellular signaling molecules (Bockaert 1999, Gether 2000, Kristiansen 2004). The GPCR superfamily participates in a variety of physiological processes such as reproduction, growth, homeostasis, metabolism, food intake, behaviors, sleep, and so on. Therefore, many members of this superfamily are major targets of pharmaceutical drugs (Wilson and Bergsma, 2000).

Rhodopsin was the first GPCR whose primary amino acid sequence and possible topological structure were identified (Nathans and Hogness, 1983). The subsequent identification of the β2‐adrenergic receptor sequence gave rise to the idea that receptors that couple to G protein share a similar seven‐helix topology (Dixon et al., 1986). This sequence information about these two receptors allowed homology‐based screening approaches such as the degenerate polymerase chain reaction (PCR) and low‐stringency hybridization, and led to the identification of new GPCR members (Bunzow 1988, Libert 1989, O'Dowd 1997). The database of expressed sequence‐tagged cDNAs (ESTs) also permitted further expansion of the GPCR superfamily (Lee 2001, Marchese 1999a), and completion of the first draft of the human genome revealed the sequences of almost all GPCRs, including those of unknown function and with unknown ligands (Lander 2001, Venter 2001). This led to efforts to reclassify the GPCR subfamily and to identify novel uncharacterized GPCRs (Fredriksson 2003a, Kristiansen 2004, Takeda 2002, Vassilatis 2003). Currently, it is thought that the human genome contains approximately 853 genes of the GPCR superfamily (Fredriksson 2005, Gloriam 2005, Young 2002). Among these, about 478 encode olfactory and gustatory GPCRs referred to as chemosensory GPCRs because they recognize signals of external origin sensed as odors, pheromones, and tastes (Young 2002, Zozulya 2001). Thus, the human genome contains approximately 375 nonchemosensory/transmitter GPCRs that bind a variety of ligands including biogenic amines, amino acids, short and long peptides, proteins, nucleotides, and lipids (Fredriksson 2003a, Vassilatis 2003).

The nonchemosensory/transmitter GPCRs include many whose endogenous ligands are unknown, the so‐called orphans (orphan GPCRs) (Marchese 1999b, Wilson 2000). Because identification of the ligands for orphan GPCRs is important for understanding the roles of these receptors, and for providing a rich source of potential drug candidates, efforts have been made to deorphan these receptors (Civelli 2005, Robas 2003a, Wise 2004). About 87 orphan GPCRs have been paired with about 70 different ligands (Table I). Most of the deorphaned receptors respond to a single compound, but some share a single ligand, or respond to more than two different compounds with different affinities (Civelli 2005, Wise 2004). Currently, about 120 orphan GPCRs remain to be deorphaned (Table I).

Various strategies have been used to increase the chances of discovering relevant ligands (Robas et al., 2003a). Classification of orphan GPCRs together with insight into the structure and function of related GPCRs may help in predicting the nature of the ligand. Knowledge of the various signal transduction pathways activated by GPCRs has led to the development of a variety of screening systems. In addition, clarification of receptor activation mechanisms as agonist dependent or agonist independent provides strategies for identifying inverse agonists and allosteric agonists. This review describes the classification of GPCRs, their activation mechanisms and associated signal transduction pathways, and current screening systems for the ligands of orphan GPCRs.

Section snippets

General Structure of GPCR Families

GPCRs share a similar topology, with seven transmembrane helices (TMHs) connected by three extracellular loops (ECLs), and three intracellular loops (ICLs); the N terminus is on the extracellular side of the membrane, and the C terminus is on the cytoplasmic side (Baldwin 1993, Donnelly 1994). Determination of the crystal structure of bovine rhodopsin showed that the polypeptide folds into seven helical segments spanning the membrane, and that these TMHs are largely α‐helical, but each is bent

GPCR Activation and G Protein Coupling

Various regulatory molecules bind to the specific domains of GPCRs. In general, small ligands, such as amines, bind primarily to the core of TMHs, middle‐size peptides to the ECLs and TMHs, and large peptides and proteins to the N termini and ECLs of their specific receptors. Ligand binding induces conformational changes of the receptors involving movement of the TMHs (Gether 1995, Schwartz 1994). Such changes are likely to induce alterations in the conformation of the ICLs, and therefore

Deorphaned GPCRs

The identification of 207 novel orphan GPCRs led to efforts to identify their ligands by various strategies. These included prediction of the ligand nature based on receptor sequence homology, choice of tissue extracts and synthetic compound libraries for ligand activity, and a variety of functional screening assays. The receptor sequence homology‐based approaches help predict the nature of the ligand for an orphan GPCR. Many GPCRs are clustered within a subfamily and have a high degree of

Concluding Remarks

Because known GPCRs have often been successfully used as therapeutic targets, orphan GPCRs may serve as a rich source of potential targets for drug discovery. Thus, many companies and academic investigators have invested heavily in the analysis of orphan GPCRs, with the aim of identifying new receptor sequences, analyzing their expression, discovering their cognate and/or surrogate ligands, and assigning the functions of novel receptor–ligand pairs. About 70 novel or known molecules such as

Acknowledgments

This study was supported by a grant (M103KV010004 03K2201 00410) from the Brain Research Center of the 21st Century Frontier Research Program. Address all correspondence to Jae Young Seong, Ph.D., Laboratory of G Protein Coupled Receptors, Korea University College of Medicine, Seoul 136‐705, Republic of Korea, Tel: +82‐2‐920‐6090, Fax: +82‐2‐921‐4355, [email protected].

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