Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectivity is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein (s) In this study, we have used the m2 and m3 muscarinic acetylcholine receptors, which selectively couple to G proteins of the G1/o and Gq family (7-10), respectively, as model systems to identify the receptor site that can functionally interact with the C terminus of GaC/o subunits.. By using a coexpression strategy involving the use of hybrid m2/m3 muscarinic receptors and C-terminally modified aq subunits, we demonstrate that a 4-aa epitope on the m2 muscarinic receptor, located at the junction between the third intracellular loop (i3 loop) and the sixth transmembrane domain (TM VI), can contact the C terminus of Gac/o subunits and that this interaction is critical for G-protein activation.MATERIALS AND METHODS DNA Constructs. The construction of pcDNA-I-based expression plasmids coding for wild-type aq (mouse) and the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.various mutant aq subunits has been described (4). Hybrid m2/m3 muscarinic receptors were prepared by standard PCRbased mutagenesis techniques (11) using Hm2pCD (encoding the human m2 receptor) and Rm3pCD (encoding the rat m3 receptor) as templates (12). The construction of CR1 and CR5 has been described previously (8
The gene encoding the murine beta 3‐adrenergic receptor (beta 3AR) has been isolated. It translates into a polypeptide of 388 amino acid residues which shows 82% overall homology with the human beta 3AR. In Southern blot experiments, a probe derived from the murine beta 3AR gene hybridizes to a unique restriction fragment in the murine and human genomes. In both species, the beta 3AR gene is located on chromosome 8, in regions (8A2‐‐‐‐8A4 in mouse, and 8p11‐‐‐‐8p12 in man) which are conserved between mouse and man. The pharmacological profile of the mouse beta 3AR strongly resembles that of the human beta 3AR. It is characterized by a low affinity toward the radiolabelled beta‐adrenergic antagonist [125I]Iodocyanopindolol and a low efficiency of other antagonists such as propranolol, ICI 118551 or CGP 20712A to inhibit cAMP production induced by isoproterenol. Another salient feature shared by the murine and the human beta 3ARs is the very potent effect of the lipolytic compound BRL 37344 on cAMP accumulation and the partial agonistic effect of the beta 1‐ and beta 2‐adrenergic antagonists CGP 12177A, oxprenolol and pindolol. These properties are very close to those ascribed to the atypical beta AR of rodent adipocytes. In addition, Northern blot analyses indicate that the beta 3AR gene is mainly expressed in mouse brown and white adipose tissues, suggesting that the murine beta 3AR described here is the atypical beta AR involved in the control of energy expenditure in fat tissue.
Each G protein-coupled receptor can interact only with a limited number of the many structurally similar G proteins expressed within a cell. This study was undertaken to identify single amino acids required for selectively coupling the m3 muscarinic acetylcholine receptor to G proteins of the Gq/11 family. To this goal, distinct intracellular segments/amino acids of the m3 receptor were systematically substituted into the structurally closely related m2 muscarinic receptor, which couples to Gi/o proteins, not Gq/11 proteins. The resultant mutant receptors were expressed in COS-7 cells and studied for their ability to induce agonist-dependent stimulation of phosphatidylinositol hydrolysis, a response known to be mediated by G proteins of the Gq/11 class. Using this approach, we were able to identify four amino acids in the second intracellular loop and four amino acids at the C terminus of the third intracellular loop of the m3 muscarinic receptor that are essential for efficient Gq/11 activation. We could demonstrate that these amino acids, together with a short segment at the N terminus of the third intracellular loop, fully account for the G protein coupling preference of the m3 muscarinic receptor. Taken together, our data strongly suggest that only a limited number of amino acids, located on different intracellular regions, are required to determine the functional profile of a given G protein-coupled receptor.
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