Parathyroid Hormone Receptor Trafficking Contributes to the Activation of Extracellular Signal-regulated Kinases but Is Not Required for Regulation of cAMP Signaling

Division of Endocrinology and Metabolism, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2005; 280(12):11281-8. DOI: 10.1074/jbc.M413393200
Source: PubMed


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.

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    • "Mechanisms accounting for PTH and PTHrP binding and activation of PTH1R have been widely investigated and support a two-site model involving initial rapid binding of the peptide C-terminal region (AAs 23-34) to the PTH1R N terminus, followed by a slower interaction of the peptide N terminus (AAs 1-14) with the J-domain containing transmembrane a-helices and interconnecting extracellular loops (Gardella and Jüppner, 2001; Castro et al., 2005; Gensure et al., 2005). Activation of PTH1R leads to regulation of multiple signaling pathways, including G s /adenylyl cyclase/cAMP/protein kinase A (PKA)-(Abou- Samra et al., 1992), G q/11 /phospholipase Cb/calcium/protein kinase C (PKC)-(Abou-Samra et al., 1992), and G 12/13 /Ras homolog gene family, member A/phospholipase D-signaling (Wang and Stern, 2010), as well as G protein–dependent and G protein – independent and b-arrestin – dependent activation of extracellular signal - related kinase ( ERK ) 1 / 2 ( Syme et al., 2005 ; Gesty - Palmer et al., 2006 ). PTH1R-mediated activation of PKC also occurs through a phospholipase Cb ( PLCb ) - independent mechanism ( Takasu et al. , 1999 ) . "
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    ABSTRACT: Parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP), acting through the osteoblast PTH1R, play important roles in bone remodeling. Intermittent administration of PTH(1-34) (teriparatide) leads to bone formation while continuous administration paradoxically leads to bone resorption. Activation of PTH1R promotes regulation of multiple signaling pathways, including Gs/cAMP/PKA, Gq/calcium/PKC, β-arrestin recruitment and ERK1/2 phosphorylation as well as receptor internalization but their role in promoting anabolic and catabolic actions of PTH(1-34) are unclear. In the present investigation, a collection of PTH(1-34) and PTHrP(1-34) peptide analogs were evaluated in orthogonal hPTH1R functional assays capturing Gs- and Gq-signaling, β-arrestin recruitment, ERK1/2 phosphorylation and receptor internalization to further define patterns of of PTH1R signaling they stimulate and to further establish peptide domains contributing to agonist activity. Results indicate that both N- and C-terminal domains are critical for activation of signaling pathways. However, modifications of both regions leads to more substantial decreases in agonist potency and efficacy to stimulate Gq-signaling, β-arrestin recruitment, ERK1/2 phosphorylation and receptor internalization than to stimulate Gs. The substantial contribution of the peptide C-terminal domain in activation of hPTH1R-signaling suggests a role in positioning of the peptide N-terminal region into the receptor J-domain. Several PTH and PTHrP peptide analogs evaluated in this study promote different patterns of biased agonist signaling and may serve as useful tools to further elucidate therapeutically relevant PTH1R signaling in osteoblasts. With a better understanding of therapeutically relevant signaling, novel biased peptides with desired signaling could be designed for safer and more effective treatment of osteoporosis.
    Preview · Article · Mar 2013 · Journal of Pharmacology and Experimental Therapeutics
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    • "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. "
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    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.
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    • "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). "
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