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Calcium-Sensing Receptor Activation of Rho Involves Filamin and Rho-Guanine Nucleotide Exchange Factor

Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: L. Darryl Quarles, M.D., Duke University Medical Center, Box 3036, Durham, North Carolina 27710. E-mail: quarl001{at}mc.duke.edu.

We investigated the role of Gq, filamin, Rho, the RhoGEF Lbc, and the C terminus of calcium-sensing receptor (CasR) in CasR signaling. We found that Ca²⁺, Mg²⁺, or the calcimimetic R isomer of N-(3-[2-chlorophenyl]propyl)-(R)--methyl-3-methoxybenzylamine (NPS-R568) stimulated serum response element (SRE) activity human embryonic kidney 293 cells transfected with CasR and an SRE-luciferase reporter construct. Coexpression of either the dominant negative Gq(305–359) minigene, regulators of G protein signaling (RGS)2 or RGS4, inhibited CasR-stimulated SRE activity, consistent with CasR activation of Gq. The cytoskeletal associated Rho protein is involved CasR activation of SRE, as evidenced by CasR-mediated increase in membrane-associated Rho A and by the ability of Clostridium botulinum C3 (C3) exoenzyme to inhibit both CasR and GqQL-stimulated SRE activity. Overexpression of the RhoGEF Lbc, lacking either the Dbl-homology or Pleckstrin homology domain, as well as the filamin peptide (1530–1875) inhibited CasR-mediated activation of SRE. A carboxyl-terminal CasR minigene, CasR(906–980), encoding a filamin binding region, also blocked CasR- and GqQL-stimulated SRE activity. Potential interactions between CasR, RhoGEF Lbc, Rho A, Gq, and filamin were demonstrated by reciprocal coimmunoprecipitation studies. Our results suggest that the C terminus of CasR may interact with filamin to create a cytoskeletal scaffold necessary for the spatial organization of Gq, RhoGEF Lbc, and Rho signaling pathways upstream of SRE activation.