The type I PTH/PTH-related peptide receptor (PTH1R), 2 a seventransmembrane receptor (7TMR) highly expressed in the kidney and bone, plays a fundamental role in the regulation of calcium homeostasis, as well as in bone formation and resorption. Ligands for PTH1R including PTHrp and PTH are involved in the etiology and treatment of disease processes such as hypercalcemia of malignancy and osteoporosis. The actions of PTH, however, are complex. PTH is known for both anabolic and catabolic effects on bone, which are dependent upon intermittent or persistent exposure, respectively (1-3). The mechanistic basis of these effects on bone remodeling are not well understood.The intracellular signaling pathways activated by PTH and PTHrP via the PTH1R receptor include G s -mediated activation of adenylate cyclase, resulting in cAMP production and PKA activation, and G q/11 -mediated PLC stimulation, leading to inositol 1,4,5-trisphosphate (IP 3 ) production, calcium mobilization, and PKC activation (4 -7). It has also been demonstrated that PTH activates the Raf-MEK-ERK MAP kinase (MAPK) cascade through both PKA and PKC in a cell-specific and G protein-dependent manner (8 -10). MAPKs activated in response to stimulation by many different classes of cell surface receptors, including growth factor receptor tyrosine kinases and 7TMRs, regulate cell growth, division, differentiation, and apoptosis (11). PTHstimulated activation of MAPK is known to have proliferative effects in kidney and bone (12, 13).There is growing evidence for novel 7TMR signaling mechanisms that are distinct from the classical G protein second messenger-dependent pathways. One such mechanism involves -arrestins, a small family of cytosolic proteins initially identified for their central role in 7TMR desensitization. -Arrestins are recruited to agonist-occupied 7TMRs that have been phosphorylated by specialized G protein-coupled receptor kinases (GRKs) and sterically inhibit receptor-G protein coupling resulting in homologous receptor desensitization. Additionally, -arrestins act as adaptors in clathrin-mediated receptor endocytosis (14, 15). The role of -arrestins acting as signal transducers through the formation of scaffolding complexes with accessory effector molecules such as Src, Ras, ERK1/2, JNK3, and MAPK kinase 4 (MKK4) is becoming increasingly recognized (16 -20).The potential signaling diversity of 7TMRs suggests the possible existence of multiple discrete "active" receptor conformations. This implies that specific ligands might direct distinct signaling responses by preferentially stabilizing one or more of these active conformations. In the simple two-state model of receptor activation, agonists are defined as drugs that stabilize the active receptor conformation, which in turn promotes G protein activation. Conversely, an inverse agonist preferentially binds to the inactive receptor conformational state thereby reduc-
Angiotensin II, acting through type 1 angiotensin (AT 1 ) receptors, has potent effects that alter renal excretory mechanisms. Control of sodium excretion by the kidney has been suggested to be the critical mechanism for blood pressure regulation by the renin-angiotensin system (RAS). However, since AT 1 receptors are ubiquitously expressed, precisely dissecting their physiological actions in individual tissue compartments including the kidney with conventional pharmacological or gene targeting experiments has been difficult. Here, we used a cross-transplantation strategy and AT 1A receptor-deficient mice to demonstrate distinct and virtually equivalent contributions of AT 1 receptor actions in the kidney and in extrarenal tissues to determining the level of blood pressure. We demonstrate that regulation of blood pressure by extrarenal AT 1A receptors cannot be explained by altered aldosterone generation, which suggests that AT 1 receptor actions in systemic tissues such as the vascular and/or the central nervous systems make nonredundant contributions to blood pressure regulation. We also show that interruption of the AT 1 receptor-mediated short-loop feedback in the kidney is not sufficient to explain the marked stimulation of renin production induced by global AT 1 receptor deficiency or by receptor blockade. Instead, the renin response seems to be primarily determined by renal baroreceptor mechanisms triggered by reduced blood pressure. Thus, the regulation of blood pressure by the RAS is mediated by AT 1 receptors both within and outside the kidney.
About 40% of the therapeutic agents in use today exert their effects through seven-transmembrane receptors (7TMRs). When activated by ligands, these receptors trigger two pathways that independently transduce signals to the cell: one through heterotrimeric GTP-binding proteins (G proteins) and one through β-arrestins; so-called biased agonists can selectively activate these distinct pathways. Here, we investigate selective activation of these pathways through the use of a biased agonist for the type 1 parathyroid hormone (PTH)-PTH-related protein receptor (PTH1R), (DTrp 12 , Tyr 34 )-PTH(7-34) (PTH-βarr), which activates β-arrestin but not classic G protein signaling. In mice, PTH-βarr induces anabolic bone formation, as does the nonselective agonist PTH (1-34), which activates both mechanisms. In β-arrestin2-null mice, the increase in bone mineral density evoked by PTH(1-34) is attenuated and that stimulated by PTH-βarr is ablated. The β-arrestin2-dependent pathway contributes primarily to trabecular bone formation and does not stimulate bone resorption. These results show that a biased agonist selective for the β-arrestin pathway can elicit a response in vivo distinct from that elicited by nonselective agonists. Ligands with these properties may form the basis for improved 7TMR-directed pharmacologic agents with enhanced therapeutic specificity.
The Na؉ /H ؉ exchanger regulatory factor (NHERF) is constitutively phosphorylated in cells, but the site(s) of this phosphorylation and the kinase(s) responsible for it have not been identified. We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK-293) cells is Ser 289 , and that the stoichiometry of phosphorylation is near 1 mol/mol. NHERF contains two PDZ domains that recognize the sequence S/T-X-L at the carboxyl terminus of target proteins, and thus we examined the possibility that kinases containing this motif might associate with and phosphorylate NHERF. Overlay experiments and co-immunoprecipitation studies revealed that NHERF binds with high affinity to a splice variant of the G protein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R-L. NHERF does not associate with GRK6B or GRK6C, alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini. GRK6A phosphorylates NHERF efficiently on Ser 289 in vitro, whereas GRK6B, GRK6C, and GRK2 do not. Furthermore, the endogenous "NHERF kinase" activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction and that GRK6A is the kinase in HEK-293 cells responsible for the constitutive phosphorylation of NHERF.
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