Characterization of the extra-large G protein alpha-subunit XL alpha s - II. Signal transduction properties

In the preceding paper (Pasolli, H. A., Klemke, M., Kehlenbach, R. H., Wang , Y., and Huttner, W. B. (2000) J. Biol. Chem. 275, 33622-33632), we report on the tissue distribution and subcellular localization of XL alphas (extr a large alphas), a neuroendocrine-specific, plasma membrane-associated prot ein consisting of a novel 37-kDa XL domain followed by a 41-kDa alphas doma in encoded by exons 2-13 of the G alphas gene. Here, we have studied the si gnal transduction properties of XL alphas. Like G alphas, XL alphas undergo es a conformational change upon binding of GTP gammaS (guanosine 5'-O-(thio )triphosphate), as revealed by its partial resistance to tryptic digestion, which generated the same fragments as in the case of G alphas. Two approac hes were used to analyze XL alphas-beta gamma interactions: (i) ADP-ribosyl ation by cholera toxin to detect even weak or transient XL alphas-beta gamm a interactions and (ii) sucrose density gradient centrifugation to reveal s table heterotrimer formation. The addition of beta gamma subunits resulted in an increased ADP-ribosylation of XL alphas as well as an increased sedim entation rate of XL alphas in sucrose density gradients, indicating that XL alphas interacts with the beta gamma dimer. Surprisingly, however, XL alph as, in contrast to G alphas, was not activated by the beta2-adrenergic rece ptor upon reconstitution of S49cyc(-) membranes. Similarly, using photoaffi nity labeling of pituitary membranes with azidoanilide-GTP, XL alphas was n ot activated upon stimulation of pituitary adenylyl cyclase-activating poly peptide (PACAP) receptors or other G alphas-coupled receptors known to be p resent in these membranes, whereas G alphas was. Despite the apparent inabi lity of XL alphas to undergo receptor-mediated activation, XL alphas-GTP ga mmaS markedly stimulated adenylyl cyclase in S49cyc(-) membranes. Moreover, transfection of PC12 cells with a GTPase-deficient mutant of XL alphas, XL alphas-Q548L, resulted in a massive increase in adenylyl cyclase activity. Our results suggest that in neuroendocrine cells, the two related G protei ns, G alphas and XL alphas, exhibit distinct properties with regard to rece ptor-mediated activation but converge onto the same effector system, adenyl yl cyclase.