Many of the G-protein-coupled receptors for hormones that bind to the cell surface can signal to the interior of the cell through several different classes of G protein. For example, although most of the actions of the prototype beta2-adrenergic receptor are mediated through Gs proteins and the cyclic-AMP-dependent protein kinase (PKA) system, beta-adrenergic receptors can also couple to Gi proteins. Here we investigate the mechanism that controls the specificity of this coupling. We show that in HEK293 cells, stimulation of mitogen-activated protein (MAP) kinase by the beta2-adrenergic receptor is mediated by the betagamma subunits of pertussis-toxin-sensitive G proteins through a pathway involving the non-receptor tyrosine kinase c-Src and the G protein Ras. Activation of this pathway by the beta2-adrenergic receptor requires that the receptor be phosphorylated by PKA because it is blocked by H-89, an inhibitor of PKA. Additionally, a mutant of the receptor, which lacks the sites normally phosphorylated by PKA, can activate adenylyl cyclase, the enzyme that generates cAMP, but not MAP kinase. Our results demonstrate that a mechanism previously shown to mediate uncoupling of the beta2-adrenergic receptor from Gs and thus heterologous desensitization (PKA-mediated receptor phosphorylation), also serves to 'switch' coupling of this receptor from Gs to Gi and initiate a new set of signalling events.

(maximum stimulation Ϸ50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular ␤-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1͞2 activation with Ϸ60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular ␤-arrestin 2 by small interfering RNA (␤-arrestin dependent). These findings imply the existence of independent G protein-and ␤-arrestin 2-mediated pathways leading to ERK1͞2 activation and the existence of distinct ''active'' conformations of a seven-membrane-spanning receptor coupled to each.

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of ␤-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinintagged AT1aR, angiotensin stimulation triggered ␤-arrestin-2 binding to the receptor and internalization of AT1aR-␤-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-␤-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, ␤-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged ␤-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with ␤-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with ␤-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to ␤-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in ␤-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to ␤-arrestin-2, and the association of ␤-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that ␤-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

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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-

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.

Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.