Extralarge XLαs (XXLαs), a Variant of Stimulatory G Protein α-Subunit (Gsα), Is a Distinct, Membrane-Anchored GNAS Product that Can Mimic Gsα

GNAS gives rise to multiple imprinted gene products, including the alpha-subunit of the stimulatory G protein (Gs alpha) and its variant XL alpha s. Based on genomic sequence, the translation of XL alpha s begins from the middle of a long open reading frame, suggesting the existence of an N-terminally extended variant termed extralarge XL alpha s (XXL alpha s). Although XXL alpha s, like Gs alpha and XL alpha s, would be affected by most disease-causing GNAS mutations, its authenticity and biological significance remained unknown. Here we identified a mouse cDNA clone that comprises the entire open reading frame encoding XXL alpha s. Whereas XXL alpha s mRNA was readily detected in mouse heart by RT-PCR, it appeared virtually absent in insulinoma-derived INS-1 cells. By Northern blots and RT-PCR, XXL alpha s mRNA was detected primarily in the mouse brain, cerebellum, and spleen. Immunohistochemistry using a specific anti-XXL alpha s antibody demonstrated XXL alpha s protein in multiple brain areas, including dorsal hippocampus and cortex. In transfected cells, full-length human XXL alpha s was localized to the plasma membrane and mediated isoproterenol-and cholera toxin-stimulated cAMP accumulation. XXL alpha s-R844H, which bears a mutation analogous to that in the constitutively active Gs alpha mutant Gs alpha-R201H (gsp oncogene), displayed elevated basal signaling. However, unlike Gs alpha-R201H, which mostly remains in the cytoplasm, both XXL alpha s-R844H and a constitutively active XL alpha s mutant localized to the plasma membrane. Hence, XXL alpha s is a distinct GNAS product and can mimic Gs alpha,but the constitutively active XXL alpha s and Gs alpha mutants differ from each other regarding subcellular targeting. Our findings suggest that XXL alpha s deficiency or hyperactivity may contribute to the pathogenesis of diseases caused by GNAS mutations. (Endocrinology 150: 3567-3575, 2009)