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Extralarge XLs (XXLs), a Variant of Stimulatory G Protein -Subunit (Gs), Is a Distinct, Membrane-Anchored GNAS Product that Can Mimic Gs

Endocrine Unit (C.A., M.J.M., H.A.W.T., M.B.), Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; Department of Endodontics (C.A.), Center for Dental Sciences, Gülhane School of Medicine, 06018 Ankara, Turkey; Departments of Neurology (N.A., N.W.K., A.D.) and Pathology (N.W.K.), Boston University School of Medicine, Boston, Massachusetts 02115; and Research and Development Service/Geriatric Research Education and Clinical Center (N.A., N.W.K., A.D.), Veterans Affairs Boston Healthcare System, Boston, Massachusetts 02130

Address all correspondence and requests for reprints to: Murat Bastepe, M.D., Ph.D., Endocrine Unit, Massachusetts General Hospital, 50 Blossom Street, Thier 10, Boston, Massachusetts 02114. E-mail: bastepe{at}helix.mgh.harvard.edu.

GNAS gives rise to multiple imprinted gene products, including the -subunit of the stimulatory G protein (Gs) and its variant XLs. Based on genomic sequence, the translation of XLs begins from the middle of a long open reading frame, suggesting the existence of an N-terminally extended variant termed extralarge XLs (XXLs). Although XXLs, like Gs and XLs, 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 XXLs. Whereas XXLs 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, XXLs mRNA was detected primarily in the mouse brain, cerebellum, and spleen. Immunohistochemistry using a specific anti-XXLs antibody demonstrated XXLs protein in multiple brain areas, including dorsal hippocampus and cortex. In transfected cells, full-length human XXLs was localized to the plasma membrane and mediated isoproterenol- and cholera toxin-stimulated cAMP accumulation. XXLs-R844H, which bears a mutation analogous to that in the constitutively active Gs mutant Gs-R201H (gsp oncogene), displayed elevated basal signaling. However, unlike Gs-R201H, which mostly remains in the cytoplasm, both XXLs-R844H and a constitutively active XLs mutant localized to the plasma membrane. Hence, XXLs is a distinct GNAS product and can mimic Gs, but the constitutively active XXLs and Gs mutants differ from each other regarding subcellular targeting. Our findings suggest that XXLs deficiency or hyperactivity may contribute to the pathogenesis of diseases caused by GNAS mutations.