High specificity of human orexin receptors for orexins over neuropeptide Y and other neuropeptides.

Orexin- and neuropeptide Y-ergic systems show physiological interaction in the regulation of appetite. In this study we investigate the postulated effect of neuropeptide Y (NPY) and other peptides directly on orexin OX1 and OX2 receptors. None of the tested peptides (NPY-variants, secretin, alpha-melanocortin, pancreatic polypeptide or pituitary adenylyl cyclase-activating peptide-38) induced any Ca2+ elevation at the concentrations up to 300 nM (human NPY, secretin and alpha-melanocortin up to 1 microM). Orexin-A- and -B-mediated Ca2+ elevations were completely unaffected by the peptides. In binding assays, human NPY, secretin and alpha-melanocortin at 1 microM did not induce any displacement of 0.1 nM [125I]orexin-A. Thus, in contrast to the previously reported result on orexin-A binding, our results demonstrate that NPY does not directly interact with orexin receptor in intact cellular settings.

Different domains in the third intracellular loop of the GLP-1 receptor are responsible for Galpha(s) and Galpha(i)/Galpha(o) activation.

It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Galpha(s) proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Galpha(s) but also to the Galpha(i)/Galpha(o) type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.