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 ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steadystate levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.Type II diabetes mellitus is a complex metabolic disease with an environmental and genetic component. Hyperglycemia develops for reasons that are not completely understood; however, a prominent defect is the inability of insulin to inhibit hepatic glucose production (HGP).1 Increased gluconeogenesis is believed to be the major cause of increased HGP and fasting hyperglycemia in type II diabetes mellitus patients (1). Gluconeogenesis is a highly regulated process, catalyzed by several enzymes subject to regulation by insulin. The first site of regulation, the conversion of oxalacetate to phosphoenolpyruvate, is catalyzed by the enzyme phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is considered a key rate-controlling enzyme in the pathway of gluconeogenesis from pyruvate, lactate, and alanine (2). Normally, insulin rapidly and substantially inhibits PEPCK gene transcription and gluconeogenesis in liver and in rat hepatoma cells. However, in several animal models of obesity and type II diabetes, gluconeogenesis and PEPCK mRNA are increased by 2-3-fold over non-diabetic animals, despite circulating insulin levels that may be ...

Inactivating mutations in distinct G protein‐coupled receptors (GPCRs) are currently being identified as the cause of a steadily growing number of human diseases. Based on previous studies showing that GPCRs are assembled from multiple independently stable folding units, we speculated that such mutant receptors might be functionally rescued by ‘supplying’ individual folding domains that are lacking or misfolded in the mutant receptors, by using a co‐expression strategy. To test the feasibility of this approach, a series of nine mutant V2 vasopressin receptors known to be responsible for X‐linked nephrogenic diabetes insipidus were used as model systems. These mutant receptors contained nonsense, frameshift, deletion or missense mutations in the third intracellular loop or the last two transmembrane helices. Studies with transfected COS‐7 cells showed that none of these mutant receptors, in contrast to the wild‐type V2 receptor, was able to bind detectable amounts of the radioligand, [3H]arginine vasopressin, or to activate the G(S)/adenylyl cyclase system. Moreover, immunological studies demonstrated that the mutant receptors were not trafficked properly to the cell surface. However, several of the nine mutant receptors regained considerable functional activity upon co‐expression with a C‐terminal V2 receptor peptide spanning the sequence where the various mutations occur. In many cases, the restoration of receptor activity by the co‐expressed receptor peptide was accompanied by a significant increase in cell surface receptor density. These findings may lead to the design of novel strategies in the treatment of diseases caused by inactivating mutations in distinct GPCRs.

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.