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J. Biol. Chem., Vol. 281, Issue 50, 38181-38188, December 15, 2006

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Proline-rich Motifs in the Parathyroid Hormone (PTH)/PTH-related Protein Receptor C Terminus Mediate Scaffolding of c-Src with -Arrestin2 for ERK1/2 Activation^*

From the Service of Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital, 1211 Geneva 14, Switzerland

Parathyroid hormone (PTH) stimulates ERK1/2 through both G-protein signaling and -arrestin2-mediated internalization. -Arrestin may serve as a scaffold for c-Src. However, the molecular mechanisms for ERK1/2 activation by PTH remain unclear. By using a targeted mutagenesis approach, we investigated the PTH/PTH-related protein receptor (PTH1R) structural determinants for ERK1/2 activation and transcriptional activity in HEK-293 cells. First, ERK1/2 activation was inhibited by PTH1R mutations that specifically abrogate G_q-protein kinase C signaling without a decrease in cAMP-protein kinase A. Second, PTH1R C-terminal mutations and/or deletions that prevent interaction with -arrestin inhibited ERK1/2 activation. Similar results were obtained in HEK-293 cells co-expressing wild-type PTH1R and a dominant-negative -arrestin2. Third, the c-Src inhibitor PP2 and a kinasedead c-SrcK295M mutant co-expressed with wild-type PTH1R both inhibited ERK1/2 activation. Furthermore, c-Src co-precipitated with both PTH1R and-arrestin2 in response to PTH. Deleting the PTH1R-proximal C terminus abolished these interactions. However, the need for receptor interaction with -arrestin to co-precipitate Src and activate ERK1/2 was obviated by expressing a constitutively active c-SrcY527A mutant, suggesting direct binding of activated Src to PTH1R. Subsequently, we identified and mutated to alanine four proline-rich motifs in the PTH1R distal C terminus, which resulted in loss of both c-Src and arrestin co-precipitation and significantly decreased ERK1/2 activation. These data delineate the multiple PTH1R structural determinants for ERK1/2 activation and newly identify a unique mechanism involving proline-rich motifs in the receptor C terminus for reciprocal scaffolding of c-Src and -arrestin2 with a class II G-protein-coupled receptor.

Received for publication, July 17, 2006 , and in revised form, September 13, 2006.

^* This work was supported by Swiss National Science Foundation Grants PPOOB-106742 (to S. L. F.) and 3100AO-112146 (to J. C.) and by the Theodor Naegeli and Julius Thorn Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 41-22-382-99-52; Fax: 41-22-382-99-73; E-mail: Serge.Ferrari{at}medecine.unige.ch.

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