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J. Biol. Chem., Vol. 279, Issue 11, 10060-10069, March 12, 2004

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Calcium-sensing Receptor-mediated ERK1/2 Activation Requires G_i2 Coupling and Dynamin-independent Receptor Internalization^*

Deborah M. Holstein, Kelly A. Berg, L. M. Fredrik Leeb-Lundberg¶, Merle S. Olson, and Christine Saunders||**

From the ^||Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600 and Departments of Biochemistry and Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900

The calcium-sensing receptor (CaR) recently has been shown to activate MAP kinase (ERK1/2) in various cell types as well as in heterologous expression systems. In this study we show that the CaR agonist NPS R-467 (1 µM), which does not activate the CaR by itself, robustly activates ERK1/2 in the presence of a low concentration of Ca²⁺ (0.5 mM CaCl₂) in human embryonic kidney (HEK) cells permanently expressing the human CaR (HEK-hCaR). Ca²⁺ (4 mM) also activates ERK1/2 but with differing kinetics. CaR-dependent ERK1/2 activation begins to desensitize to 4 mM Ca²⁺ after 10 min, whereas there is no desensitization to NPS R-467/CaCl₂ as late as 4 h. Moreover, recovery from desensitization occurs as rapidly as 30 min with 4 mM CaCl₂. Pretreatment of HEK-hCaR cells with concanavalin A (250 µg/ml) to block CaR internalization completely eliminated the NPS R-467/CaCl₂-mediated ERK1/2 activation but did not block the 2-min time point of 4 mM Ca²⁺-mediated ERK1/2 activation. Neither dominant-negative dynamin (K44A) nor dominant-negative -arrestin inhibited ERK1/2 activation by either CaR agonist treatment, suggesting that CaR-elicited ERK1/2 signaling occurs via a dynamin-independent pathway. Pertussis toxin pretreatment partially attenuated the 4 mM Ca²⁺-ERK1/2 activation; this attenuated activity was completely restored by co-expression of the G_i2 ^C351I but not G_i1 ^C351I or G_i3 ^C351I G proteins, PTX-insensitive G protein mutants. Taken together, these data suggest that both 4 mM Ca²⁺ and NPS R-467/CaCl₂ activate ERK1/2 via distinguishable pathways in HEK-hCaR cells and may represent a nexus to differentially regulate differentiation versus proliferation via CaR activation.

Received for publication, November 3, 2003 , and in revised form, November 25, 2003.

^* This work was supported by American Heart Association Grant AHA-TX 0060030Y (to C. S.) and United States Public Health Service Grant DK-02852 (to C. S.). 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.

^¶ Current address: Division of Molecular Neurobiology, Wallenberg Neuroscience Center, Lund University, BMC, A12, SE-22184 Lund, Sweden.

^** To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, 417B Preston Research Bldg., Nashville, TN 37232-6600. Tel.: 615-936-3771; Fax: 615-322-6379; E-mail: christine.saunders{at}vanderbilt.edu.

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