Extreme C Terminus of G Protein -Subunits Contains a Site That Discriminates between G i -coupled Metabotropic Glutamate Receptors* (Received for publication, May 12, 1998, and in revised form, July 9, 1998) Jaroslav Blahos II, Sophie Mary, Julie Perroy, Cyril de Colle, Isabelle Brabet, Joe ¨ l Bockaert, and Jean-Philippe Pin‡ From the Me ´canismes Mole ´culaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, F-34094 Montpellier Cedex 5, France Metabotropic glutamate receptors (mGlu receptors), the Ca 2 -sensing receptor, -aminobutyric acid type B receptors, and one group of pheromone receptors con- stitute a unique family (also called family 3) of heptahe- lical receptors. This original family shares no sequence similarity with any other G protein-coupled receptors. The identification and comparison of the molecular de- terminants of receptor/G protein coupling within the different receptor families may help identify general rules involved in this protein/protein interaction. In or- der to detect possible contact sites important for cou- pling selectivity between family 3 receptors and the G protein -subunits, we examined the coupling of the cyclase-inhibiting mGlu2 and mGlu4 receptors to chi- meric  q -subunits bearing the 5 extreme C-terminal amino acid residues of either G i ,G o , or G z . Whereas mGlu4 receptor activated all three chimeric G proteins, mGlu2 receptor activated G qi and G qo but not G qz . The mutation of isoleucine 4 of G qz into cysteine was sufficient to recover coupling of the mutant G protein to mGlu2 receptor. Moreover, the mutation of cysteine 4 of G qo into isoleucine was sufficient to suppress the coupling to mGlu2 receptor. Mutations at positions 5 and 1 had an effect on coupling efficiency, but not selectivity. Our results emphasize the importance of the residue 4 of the -subunits in their specific interaction to heptahelical receptors by extending this finding on the third family of G protein-coupled receptors. Transduction of extracellular signals to intracellular re- sponses via G protein-coupled receptors (GPCRs) 1 includes ac- tivation of the receptor and subsequent regulation of effectors via trimeric G proteins. A variety of heterotrimeric G proteins have different target proteins (1). Accordingly, the intracellular response depends on the G protein subtypes (among those located in the proximity of the receptor) that can be activated by the receptor (selectivity). Identification of the molecular basis of receptor/G protein coupling selectivity is therefore of interest since this determines the transduction mechanism of these receptors. Three major families of GPCRs can be defined, and the members of each family share no sequence similarity with receptors from the other families. Receptors homologous to rhodopsin (receptors for catecholamines, acetylcholine, certain peptides, and glycoproteins) constitute the first family (family 1) which is to date the best characterized one. Family 2 recep- tors are those homologous to the vasoactive intestinal peptide and the glucagon receptors. Family 3 receptors comprise the metabotropic glutamate receptors (mGlu receptors) (2), the -aminobutryric acid, type B receptors (3), the Ca 2+ -sensing receptor (4), and a recently discovered new family of putative pheromone receptors (5, 6). The unique feature of family 3 GPCRs is a large extracellular N terminus that forms the ligand binding site (7, 8). Common to all three families are some structural elements including seven transmembrane do- mains so that all GPCRs are also called heptahelical receptors. Molecular determinants involved in the coupling between family 1 GPCRs and G proteins have been studied (9 –11). Specific motifs in the third intracellular loop (in most cases) and the second intracellular loop (in some cases) of these re- ceptors have been shown to determine which G protein sub- types will be activated. Although the receptors probably con- tact both the -subunit, and the  dimer of the G protein (12–14), the direct interaction with the  subunit is presumed to play a critical role in the coupling specificity. On the G protein -subunit several elements have been shown to deter- mine specificity of coupling to family 1 receptors (9, 10, 15–20). Among these, the extreme C terminus was shown to play a critical role by interacting with certain motifs within a cavity formed by the second and third intracellular loops of the recep- tor (9, 11, 20 –23). Comparison of the molecular determinants of receptor/G pro- tein coupling between members of the different GPCR families should help to establish some general principles for this inter- action. Accordingly, we recently started to identify the struc- tural determinants of G protein coupling in mGlu receptors that are used here as representatives of the family 3 GPCRs. Three groups of mGlu receptors have been defined based on their sequence similarity and G protein-coupling selectivity (2, 24). In these receptors, the first and third intracellular loops (i1 and i3) are short with low variability. Major sequence differ- ences are found in the second intracellular loop (i2). The highly variable intracellular C termini play a role in G protein inter- action but probably not in a discriminatory manner (25–28). * This work was supported by grants from the CNRS, the European Community (Biomed2 (BMH4-CT96-0228) and Biotech2 (BIO4-CT96- 0049) programs), the “action incitative” “Physique et Chimie du Vivant” from the French government (PCV97-115), the Fondation pour la Re- cherche Me ´dicale, the Direction des Recherches et Etudes Techniques (DRET 91/161), and the Bayer Company (France and Germany) (to J.-P. P. and J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: Jean-Philippe Pin, Me ´canismes Mole ´culaires des Communications Cellulaires, CNRS- UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier Cedex 5, France. Tel.: 33 467 14 2933; Fax: 33 467 54 2432; E-mail: pin@ ccipe.montp.inserm.fr. 1 The abbreviations used are: GPCR, G protein-coupled receptor; mGlu, metabotropic glutamate; HEK, human embryonic kidney; IP, inositol phosphate. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 40, Issue of October 2, pp. 25765–25769, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 25765 by guest on May 30, 2020 http://www.jbc.org/ Downloaded from