Assembly of fully functional GABABreceptors requires heteromerization of the GABAB(1)and GABAB(2)subunits. It is thought that GABAB(1)and GABAB(2)undergo coiled-coil dimerization in their cytoplasmic C termini and that assembly is necessary to overcome GABAB(1)retention in the endoplasmatic reticulum (ER). We investigated the mechanism underlying GABAB(1)trafficking to the cell surface. We identified a signal, RSRR, proximal to the coiled-coil domain of GABAB(1)that when deleted or mutagenized allows for surface delivery in the absence of GABAB(2). A similar motif, RXR, was recently shown to function as an ER retention/retrieval (ERR/R) signal in KATPchannels, demonstrating that G-protein-coupled receptors (GPCRs) and ion channels use common mechanisms to control surface trafficking. A C-terminal fragment of GABAB(2)is able to mask the RSRR signal and to direct the GABAB(1)monomer to the cell surface, where it is functionally inert. This indicates that in the heteromer, GABAB(2)participates in coupling to the G-protein. Mutagenesis of the C-terminal coiled-coil domains in GABAB(1)and GABAB(2)supports the possibility that their interaction is involved in shielding the ERR/R signal. However, assembly of heteromeric GABABreceptors is possible in the absence of the C-terminal domains, indicating that coiled-coil interaction is not necessary for function. Rather than guaranteeing heterodimerization, as previously assumed, the coiled-coil structure appears to be important for export of the receptor complex from the secretory apparatus.

Although G-protein-coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the ␥-aminobutyric acid (GABA) type B receptor (GABA B R) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit, which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into homodimers (Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., Pré zeau, L., and Pin, J. -P. (2001) EMBO J. 20, 2152-2159).In the present study, we have examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that end, point mutations in the highly conserved third intracellular loop known to prevent Gprotein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the Gprotein was assessed by measurement of inositol phosphate accumulation, or in cultured neurons where the inhibition of the Ca 2؉ channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABA B receptor either in HEK293 cells or in neurons. Accordingly, whereas in all GPCRs the same protein is responsible for both agonist binding and Gprotein activation, these two functions are assumed by two distinct subunits in the GABA B heteromer: one subunit, GB1, binds the agonists whereas the other, GB2, activates the G-protein. This illustrates the importance of a single subunit for G-protein activation within a dimeric receptor.

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G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the ␥-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of ␥-aminobutyric acid type B 2 can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation. G protein-coupled receptors (GPCRs)2 are encoded by one of the most important gene families in mammalian genomes (1). These membrane proteins play a critical role in transducing extracellular signals into the cell and constitute the major target for drug development. Although they are important molecules, little is known about their activation mechanism (2, 3). All these receptors possess a heptahelical domain (HD), the structure of which has been solved for rhodopsin only (4). This available structure is in a fully inactive state, as it is stabilized by the covalently linked inverse agonist cis-retinal. Thus, our actual knowledge on the active conformation relies mostly on functional and biophysical analysis of a variety of mutated receptors and their coupling to G proteins and their effectors (2, 3).Four main classes of GPCRs have been defined in mammals based on sequence analysis (1, 5, 6), with the rhodopsin-like class A receptors being the largest and the most studied. Class A receptor activation is associated with a movement of TM6 relative to TM3, leading to the opening of a cavity between the intracellular loops 2 and 3 connecting TM3 to TM4 and TM5 to TM6, respectively (2, 7). The inactive state is stabilized by a network of interactions between residues at the cytoplasmic end of the TMs (4). This network includes ionic interactions that involve the highly conserved class A residues (D/E)RY at the cytoplasmic end of TM3. In many class A GPCRs, the Arg of the (D/E)RY motif makes an ionic interaction with a conserved acidic residue (D/E) of TM6 (3). Mutation of these residues leads either to a loss or a gain of function, consistent with this motif being involved in a lock that can control the conformational state of class ...