Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor.
We investigated the role of Galphaq, filamin, Rho, the RhoGEF Lbc, and the C terminus of calcium-sensing receptor (CasR) in CasR signaling. We found that Ca(2+), Mg(2+), or the calcimimetic R isomer of N-(3-[2-chlorophenyl]propyl)-(R)-alpha-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 Galphaq(305-359) minigene, regulators of G protein signaling (RGS)2 or RGS4, inhibited CasR-stimulated SRE activity, consistent with CasR activation of Galphaq. 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 GalphaqQL-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 GalphaqQL-stimulated SRE activity. Potential interactions between CasR, RhoGEF Lbc, Rho A, Galphaq, 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 Galphaq, RhoGEF Lbc, and Rho signaling pathways upstream of SRE activation.
Pi
Min
M
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Spurney
Robert F
RF
Tu
Qisheng
Q
Hinson
Todd
T
Quarles
L Darryl
LD
D054758:A Kinase Anchor Proteins; D000209:Acute-Phase Proteins / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D003285:Contractile Proteins / Q000235:genetics / Q000502:physiology*; D064448:Filamins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D005799:Genes, Dominant; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000502:physiology*; D015778:Minor Histocompatibility Antigens; D010446:Peptide Fragments / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000502:physiology*; D064067:Rho Guanine Nucleotide Exchange Factors; D027602:Serum Response Element / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000502:physiology
D054758:A Kinase Anchor Proteins; C085467:AKAP13 protein, human; D000209:Acute-Phase Proteins; D048868:Adaptor Proteins, Signal Transducing; D003285:Contractile Proteins; D064448:Filamins; D020662:Guanine Nucleotide Exchange Factors; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D064067:Rho Guanine Nucleotide Exchange Factors; C010781:acute-phase protein rho; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein