Associate editor: J. WessEvolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors
Introduction
G-protein-coupled receptors (GPCRs) transmit into the cell external signals that can be as different as photons and large glycoproteins. Accordingly, these receptors play a major role in cell-cell communication, being the receptors for most hormones and neurotransmitters. They also play an important role in the perception of the environment, being activated by light, odorants, pheromones, and taste compounds. It is not surprising, therefore, that such receptors expanded during animal evolution, representing 1–2% of the total number of genes in mammals. Surprisingly, such receptors did not expand in plants where there is only one gene encoding a GPCR, and a few specific seven transmembrane domain (7TM) proteins that have been identified in the genome of Arabidopsis thaliana Plakidou-Dymock et al., 1998, Devoto et al., 1999, Ellis & Miles, 2001.
GPCRs activate intracellular heterotrimeric G-proteins by stimulating the exchange of bound GDP in the α-subunit for GTP. Binding of GTP allows the dissociation of the α?subunit from the βγ dimer, both being able to regulate the activity of target enzymes or channels responsible for the cellular response. In addition, GPCRs may also activate intracellular pathways independently of G-proteins, possibly by interacting directly with other intracellular proteins.
All known GPCRs have a common structural domain composed of 7 transmembrane helices [also called the heptahelical domain (HD)], the N- and C-termini being extra- and intracellular, respectively. In mammals, sequence comparison of this domain helped define several families of GPCRs. The first identified family is composed of rhodopsin and related receptors, such as the catecholamine receptors, many peptide receptors, glycoprotein, and olfactory receptors. The second family is composed of receptors activated by large peptides, such as secretin and glucagon. The third GPCR family was first identified when the metabotropic glutamate receptors (mGluRs) were cloned, and now contains the Ca2+-sensing receptor (CaSR), the γ-aminobutyric acid (GABA)B receptor, some pheromone receptors, and taste receptors. Additional GPCR families can also be identified, such as those composed of the frizzled and smoothened receptors or of a specific group of pheromone receptors Bockaert & Pin, 1999, Bockaert et al., 2002.
In addition to the HD, most family 3 GPCRs contain a large extracellular domain responsible for ligand recognition. The structure of this domain of the mGlu1R recently has been solved in the presence and in the absence of the agonist glutamate or the antagonist α-methyl-4-carboxy-phenylglycine (MCPG) Kunishima et al., 2000, Tsuchiya et al., 2002. This, plus the recent demonstration that these receptors function as dimers Romano et al., 1996, Galvez et al., 2001, helped fuel the formulation of a hypothesis for the activation of these large receptors. Such findings will certainly be useful for the understanding of the activation process of all GPCRs.
In the present review, the various members of the family 3 GPCRs will be described and their phylogeny discussed. Special attention will be devoted to their structure and to our current view of their activation mechanism.
Section snippets
The metabotropic glutamate receptors
GPCRs activated by glutamate were first identified in the mid 1980s as phospholipase (PL)C-coupled receptors Sladeczek et al., 1985, Nicoletti et al., 1986, Sugiyama et al., 1987. The cloning of the first cDNA encoding the mGlu1aR was reported in 1991 independently by two groups Houamed et al., 1991, Masu et al., 1991, and revealed a protein sharing no obvious sequence similarity with the rhodopsin-like GPCRs. The mGlu1aR appeared, therefore, to be the first member of a new GPCR family. This
Phylogeny of family 3 G-protein-coupled receptors
As mentioned in Section 1, all family 3 receptors contain an HD (Fig. 1) sharing no overall significant sequence similarity with the other GPCRs. However, a few of the conserved residues of rhodopsin-like receptors are also conserved within family 3 GPCRs, strongly suggesting that these two GPCR families originate from a common ancestral gene (see Section 4). In addition to the HD, most family 3 GPCRs except the retinoic acid-induced receptors, possess a large extracellular segment that can be
Comparison with rhodopsin
Family 3 GPCRs possess 7 transmembrane helices Fig. 1, Fig. 4 like any other GPCRs. These transmembrane segments are separated by short intra- and extracellular loops, always smaller than 30 residues. The longest intracellular loop is the second one, with a maximal length of 27 residues. This contrasts with many rhodopsin-like receptors that possess a large third intracellular loop.
Because of the low sequence similarity between the HD of family 3 GPCRs and that of rhodopsin-like receptors, one
A dynamic bilobate structure
Looking for proteins sharing sequence similarity with the extracellular domain of mGluRs, O'Hara et al. (1993) identified bacterial periplasmic-binding proteins. Such proteins are found in the periplasmic space of Gram-negative bacteria, and are involved in the transport of small molecules, such as amino acids, ions, sugars, or small peptides. The resolved structure of such proteins revealed a bilobate protein, each lobe being separated by a cleft where ligands bind. In the absence of ligand,
A cysteine-rich domain for what?
The VFTM is connected to the HD via an 80-residue segment containing 9 conserved cysteines called the CRD (Fig. 1). The function of this domain is not known yet. The CRD is necessary for the production and secretion of the extracellular domains of the mGlu4R and -8R, but not for the proper folding of the VFTM Peltekova et al., 2000, Tsuji et al., 2000. Another study showed that the CRD is required for function of the mGluR and CaSR (Hu et al., 2000), although it is absent in the related GABAB
A highly variable domain
The carboxyl terminal intracellular domain of family 3 GPCRs corresponds to the less-conserved region among these receptors, not only between the different subtypes within one organism, but also between orthologues. Moreover, as described in 2.1 The metabotropic glutamate receptors, 2.3 The γ-aminobutyric acid, this region is the subject of alternative splicing, as observed for mGluRs and the GABAB2 subunit, such that a single gene can generate receptors with different C-terminal tails. Common
Metabotropic glutamate receptors and the Ca2+-sensing receptor are homodimers
Recombinant and native mGluRs were found very early to migrate in sodium dodecyl sulphate gels at a much higher molecular weight, about twice that expected. This prompted Romano et al. (1996) to identify the possible large protein stably interacting with mGluRs, and allowed them to first demonstrate that the mGlu5R is a dimer. Further analysis revealed that the mGlu5R homodimerizes, but cannot interact with the mGlu1aR, and that a disulfide bond further stabilizes the dimer. Further analysis of
Role of the dimeric Venus Flytrap module
Since the cloning of the mGlu1R, the first identified member of the family 3 GPCRs, and the demonstration that the agonist-binding site was located within its large extracellular domain, investigators have been wondering how agonist binding could induce the necessary change in conformation (or stabilize the active conformation) of the HD required for G-protein activation. After the proposal that the binding domain was folded like the periplasmic-binding proteins, several hypotheses have been
Outlook
Even though the family 3 GPCRs were discovered and characterized long after the rhodopsin-like GPCRs, much has been learned on the activation process of family 3 GPCRs. Indeed, these receptors are the first GPCRs for which the crystal structure of their binding domain has been solved without and with bound agonist or antagonist. These GPCRs are the first for which there is a clear demonstration that they function as dimers. Such findings have several consequences. Firstly, these may well change
Acknowledgements
We are grateful to all our current and former colleagues and collaborators, and especially to Jaroslav Blahos, Béatrice Duthey, Julie Kniazeff, Gilles Labesse, Philippe Rondard (Montpellier, France), Anne-Sophie Bessis, and Francine Acher (Paris, France) for much helpful discussions. The authors wish to thank Drs. J. Bockaert, J. C. Bonnafous, J. Marie, B. Mouillac, and T. Durroux for constructive discussion and support, and Sylvain Chauvières for the construction of the first evolutionary
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Present address: Department of Molecular Pharmacology, CCSR Building, Room 3230.269, Campus Drive, Stanford University Medical Center, Stanford, CA 94304, USA.