The peptide hormone relaxin produces dose-dependent stimulation of adenylyl cyclase activity in rat tissues (striatum, cardiac and skeletal muscle) and the muscle tissues of invertebrates, i.e., the bivalve mollusk Anodonta cygnea and the earthworm Lumbricus terrestris, adenylyl cyclase stimulation being more marked in the rat striatum and cardiac muscle. Our studies of the type of relaxin receptor involved in mediating these actions of relaxin involved the first synthesis of peptides 619-629, 619-629-Lys(Palm), and 615-629, which are derivatives of the primary structure of the C-terminal part of the third cytoplasmic loop of the type 1 relaxin receptor (LGR7). Peptides 619-629-Lys(Palm) and 615-629 showed competitive inhibition of adenylyl cyclase stimulation by relaxin in rat striatum and cardiac muscle but had no effect on the action of relaxin in rat skeletal muscle or invertebrate muscle, which is evidence for the tissue and species specificity of their actions. On the one hand, this indicates involvement of the LGR7 receptor in mediating the adenylyl cyclase-stimulating action of relaxin in rat striatum and cardiac muscle and, on the other, demonstrates the existence of other adenylyl cyclase signal mechanisms for the actions of relaxin in rat skeletal muscle and invertebrate muscle, not involving LGR7 receptors. The adenylyl cyclase-stimulating effect of relaxin in the striatum and cardiac muscles was found to be decreased in the presence of C-terminal peptide 385-394 of the alpha(s) subunit of the mammalian G protein and to be blocked by treatment of membranes with cholera toxin. These data provide evidence that in the striatum and cardiac muscle, relaxin stimulates adenylyl cyclase via the LGR7 receptor, this being functionally linked with G(s) protein. It is also demonstrated that linkage of relaxin-activated LGR7 receptor with the G(s) protein is mediated by interaction of the C-terminal half of the third cytoplasmic loop of the receptor with the C-terminal segment of the alpha(s) subunit of the G protein.

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (Gi); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase --> Gi protein (betagamma) --> phosphatidylinositol 3-kinase --> protein kinase C (zeta?) --> Gs protein --> adenylate cyclase --> cAMP.

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Functional disturbance in the novel adenylyl cyclase signaling mechanism (ACSM) of insulin and relaxin action in rat streptozotocin (STZ) type I diabetes was studied on the basis of the authors' conception of molecular defects in hormonal signaling systems as the main causes of endocrine diseases. Studying the functional state of molecular components of the ACSM and the mechanism as a whole, the following changes were found in the skeletal muscles of diabetic rats compared with control animals: 1) increase of insulin receptor binding due to an increase in the number of insulin binding sites with high and low affinity; 2) increase of the basal adenylyl cyclase (AC) activity and the reduction of AC-activating effect of non-hormonal agents (guanine nucleotides, sodium fluoride, forskolin); 3) reduction of ACSM response to stimulatory action of insulin and relaxin; 4) decrease of the insulin-activating effect on the key enzymes of carbohydrate metabolism, glycogen synthase and glucose-6-phosphate dehydrogenase. Hence, the functional activity of GTP-binding protein of stimulatory type, AC and their functional coupling are decreased during experimental type 1 diabetes that leads to the impairment of the transduction of insulin and relaxin signals via ACSM.

The development of experimental type II diabetes mellitus in rats was accompanied by dysfunction of inhibitory and stimulatory heterotrimeric G-proteins, components of hormone-sensitive adenylate cyclase signal system. The function of inhibitory G-proteins decreased most significantly under these conditions, which is seen from weakened regulatory effects of somatostatin (in the myocardium) and bromocriptine (in the brain striatum) realized via inhibitory G-proteins in diabetic rats compared to controls. These hormones produce less pronounced inhibitory effect on forskolin-induced activation of adenylate cyclase. In the myocardium of diabetic rats, the stimulatory effects of isoproterenol and relaxin on adenylate cyclase realized via stimulatory G-proteins were decreased to a lesser extent. In the striatum of diabetic rats the stimulatory effect of serotonin and relaxin did not differ from the control. Therefore, dysfunction of stimulatory G-proteins during type II diabetes mellitus is characterized by tissue specificity. Synthetic peptides corresponding to functionally important regions in a-subunits of G-proteins and relaxin receptor LGR7 less effectively inhibited hormone signal transduction via the adenylate cyclase system in rats with type II diabetes. These changes reflect abnormal coupling between receptors and G-proteins in tissues of diabetic rats.