Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both G(s)- and G(q)-mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and beta-arrestin1 (beta-arrestin1-(319-418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signaling. PTH-(1-34) induced rapid and robust activation of ERK1/2. A PTHrP-based analog ([p-benzoylphenylalanine1, Ile5,Arg(11,13),Tyr36]PTHrP-(1-36)NH2), which selectively activates the G(s)/cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1-34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7-34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and G(q) (but not G(s)) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor.
"Indeed, this βarrestin mutant might inhibit or not PTHR1 internalization depending on the cell context (Sneddon and Friedman, 2007;Syme et al., 2005). In addition, βarrestin 319–418 blocks PTHinduced ERK activation (Syme et al., 2005). In OB-6 osteoblastic cells, we found that this dominant negative reverses the effect of endogenous βarrestin by conferring responsiveness to PTH in the absence of Cx43. "
[Show abstract][Hide abstract] ABSTRACT: Parathyroid hormone (PTH) promotes osteoblast survival through a mechanism that depends on cAMP-mediated signaling downstream of the G protein-coupled receptor PTHR1. We present evidence herein that PTH-induced survival signaling is impaired in cells lacking connexin43 (Cx43). Thus, expression of functional Cx43 dominant negative proteins or Cx43 knock-down abolished the expression of cAMP-target genes and anti-apoptosis induced by PTH in osteoblastic cells. In contrast, cells lacking Cx43 were still responsive to the stable cAMP analog dibutyril-cAMP. PTH survival signaling was rescued by transfecting wild type Cx43 or a truncated dominant negative mutant of βarrestin, a PTHR1-interacting molecule that limits cAMP signaling. On the other hand, Cx43 mutants lacking the cytoplasmic domain (Cx43(Δ245)) or unable to be phosphorylated at serine 368 (Cx43(S368A)), a residue crucial for Cx43 trafficking and function, failed to restore the anti-apoptotic effect of PTH in Cx43-deficient cells. In addition, overexpression of wild type βarrestin abrogated PTH survival signaling in Cx43-expressing cells. Moreover, βarrestin physically associated in vivo to wild type Cx43 and to a lesser extent to Cx43(S368A) ; and this association and the phosphorylation of Cx43 in serine 368 were reduced by PTH. Furthermore, induction of Cx43(S368) phosphorylation or overexpression of wild type Cx43, but not Cx43(Δ245) or Cx43(S368A) , reduced the interaction between βarrestin and the PTHR1. These studies demonstrate that βarrestin is a novel Cx43-interacting protein and suggest that, by sequestering βarrestin, Cx43 facilitates cAMP signaling, thereby exerting a permissive role on osteoblast survival induced by PTH.
"In the case of b 2 -adrenergic receptor, the b-arrestin that binds to activated receptors recruits c-Src and initiates a second wave of signal transduction to activate ERK (Luttrell et al. 1999). While some GPCRs activate ERKs in an endocytosis-dependent manner, like the b 2 adrenergic receptor, activation of ERKs by some other GPCRs is independent of receptor internalization (Budd et al. 1999; DeGraff et al. 1999; Vogler et al. 1999; Shah et al. 2002; Syme et al. 2005). Stimulation of endogenous H2R in hippocampal CA3 pyramidal cells has been shown to activate ERK (Giovannini et al. 2003). "
[Show abstract][Hide abstract] ABSTRACT: Histamine H2 receptor (H2R) is a member of G protein-coupled receptor family. Agonist stimulation of H2R results in several cellular events including activation of adenylate cyclase and phospholipase C, desensitization of the receptor, activation of extracellular signal-regulated kinases ERK1/2, and receptor endocytosis. In this study, we identified a GTPase dynamin as a binding partner of H2R. Dynamin could associate with H2R both in vitro and in vivo. Functional analyses using dominant-negative form of dynamin (K44E-dynamin) revealed that cAMP production and the following H2R desensitization are independent of dynamin. However, the agonist-induced H2R internalization was inhibited by co-expression of K44E-dynamin. Furthermore, activation of extracellular-signal regulated kinases ERK1/2 in response to dimaprit, an H2R agonist, was attenuated by K44E-dynamin. Although H2R with truncation of 51 amino acids at its carboxy-terminus did not internalize after agonist stimulation, it still activated ERK1/2, but the degree of this activation was less than that of the wild-type receptor. Finally, K44E dynamin did not affect ERK1/2 activation induced by internalization-deficient H2R. These results suggest that the agonist-induced H2R internalization and ERK1/2 activation are partially dynamin-dependent. Furthermore, ERK1/2 activation via H2R is likely dependent of the endocytotic process rather than dynamin itself.
Journal of Neurochemistry 09/2008; 107(1):208-17. DOI:10.1111/j.1471-4159.2008.05608.x · 4.28 Impact Factor
"Here, forskolin stimulated MEK1/2 and ERK1/2 phosphorylation and was blocked by NHERF1. GPCR mediated ERK1/2 activation involves a variety of independent, but not necessarily exclusive, mechanisms including G protein-mediated signaling, transactivation of tyrosine kinase receptors, and receptor internalization (Luttrell, 2003; Syme et al., 2005). We were able to rule out receptor transactivation and receptor internalization as mediating ERK1/2 phosphorylation. "
[Show abstract][Hide abstract] ABSTRACT: Na/H exchange regulatory factor 1 (NHERF1) is a scaffolding protein that regulates signaling and trafficking of several G protein-coupled receptors (GPCRs), including the parathyroid hormone receptor (PTH1R). GPCRs activate extracellular signal-regulated kinase (ERK)1/2 through different mechanisms. Here, we characterized NHERF1 regulation of PTH1R-stimulated ERK1/2. Parathyroid hormone (PTH) stimulated ERK1/2 phosphorylation by a protein kinase A (PKA)-dependent, but protein kinase C-, cyclic adenosine 5'-monophosphate-, and Rap1-independent pathway in Chinese hamster ovary cells stably transfected with the PTH1R and engineered to express NHERF1 under the control of tetracycline. NHERF1 blocked PTH-induced ERK1/2 phosphorylation downstream of PKA. This suggested that NHERF1 inhibitory effects on ERK1/2 occur at a postreceptor locus. Forskolin activated ERK1/2, and this effect was blocked by NHERF1. NHERF1 interacted with AKT and inhibited ERK1/2 activation by decreasing the stimulatory effect of 14-3-3 binding to B-Raf, while increasing the inhibitory influence of AKT negative regulation on ERK1/2 activation. This novel regulatory mechanism provides a new model by which cytoplasmic adapter proteins modulate ERK1/2 activation through a receptor-independent mechanism involving B-Raf.
Molecular biology of the cell 05/2008; 19(4):1637-45. DOI:10.1091/mbc.E07-11-1114 · 4.47 Impact Factor
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