Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH.

Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting "knock-in" mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1-34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.

Extralarge XL(alpha)s (XXL(alpha)s), a variant of stimulatory G protein alpha-subunit (Gs(alpha)), is a distinct, membrane-anchored GNAS product that can mimic Gs(alpha).

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 XLalphas (XXL(alpha)s). Although XXL(alpha), 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.