G protein-coupled receptor kinases (GRKs) are a family of serine/threonine protein kinases that specifically recognize agonist-occupied, activated G protein-coupled receptor proteins as substrates. Phosphorylation of an activated receptor by a GRK terminates signaling, or desensitizes, that receptor by initiating the uncoupling of the receptor from heterotrimeric G proteins. The applicants have demonstrated that one of the GRKs (betaARK1) is expressed in osteoblasts. The role of this GRK in bone biology is unknown, however. Parathyroid hormone (PTH) plays a critical role in a variety of disorders of bone and mineral metabolism. It exerts a dose-dependent effect on the skeleton; given intermittently and in low doses, it is anabolic; in larger doses, it induces bone loss. For rational use in osteoporosis therapy, PTH should be administered in a manner which avoids desensitization. In this application, the following hypotheses are proposed for testing: (1) the rapid phase of desensitization seen in response to PTH is, in part, due to phosphorylation of the PTH receptor by a GRK, such as betaARK1 (beta-adrenergic receptor kinase1); and (2) the betaARK1 gene is involved in regulating the proliferative/differentiative response to PTH in osteoblasts. To test these hypotheses, studies are described with the following Specific Aims: (1) to correlate the effects of deletion of the betaARK gene on desensitization of the adenylyl cyclase response to PTH and phosphorylation of the PTH/PTHrP receptor; (2) to evaluate the ability of betaARK1 to phosphorylate the PTH/PTHrP receptor in membranes from HEK 293 cells with overexpression of the receptor; and (3) to evaluate the effect of deletion of the betaARK1 gene on the proliferative responsiveness and differentiation paradigm of osteoblasts.
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