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Calmodulin regulates Ca2+-sensing receptor-mediated Ca2+ signaling and its cell surface expression.

ABSTRACT: The Ca(2+)-sensing receptor (CaSR) is a member of family C of the GPCRs responsible for sensing extracellular Ca(2+) ([Ca(2+)](o)) levels, maintaining extracellular Ca(2+) homeostasis, and transducing Ca(2+) signaling from the extracellular milieu to the intracellular environment. In the present study, we have demonstrated a Ca(2+)-dependent, stoichiometric interaction between CaM and a CaM-binding domain (CaMBD) located within the C terminus of CaSR (residues 871-898). Our studies suggest a wrapping around 1-14-like mode of interaction that involves global conformational changes in both lobes of CaM with concomitant formation of a helical structure in the CaMBD. More importantly, the Ca(2+)-dependent association between CaM and the C terminus of CaSR is critical for maintaining proper responsiveness of intracellular Ca(2+) responses to changes in extracellular Ca(2+) and regulating cell surface expression of the receptor.

SUBMITTER: Huang Y

PROVIDER: S-EPMC2975215 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Calmodulin regulates Ca2+-sensing receptor-mediated Ca2+ signaling and its cell surface expression.

Huang Yun Y Zhou Yubin Y Wong Hing-Cheung HC Castiblanco Adriana A Chen Yanyi Y Brown Edward M EM Yang Jenny J JJ

The Journal of biological chemistry 20100908 46

The Ca(2+)-sensing receptor (CaSR) is a member of family C of the GPCRs responsible for sensing extracellular Ca(2+) ([Ca(2+)](o)) levels, maintaining extracellular Ca(2+) homeostasis, and transducing Ca(2+) signaling from the extracellular milieu to the intracellular environment. In the present study, we have demonstrated a Ca(2+)-dependent, stoichiometric interaction between CaM and a CaM-binding domain (CaMBD) located within the C terminus of CaSR (residues 871-898). Our studies suggest a wra ...[more]

PMID: 20826781

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Project description:Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS). NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1? (IL-1?) by activating caspase-1. Although several models for inflammasome activation, such as K(+) efflux, generation of reactive oxygen species and lysosomal destabilization, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. Here we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular Ca(2+) and decreased cellular cyclic AMP (cAMP). Ca(2+) or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyses inositol-1,4,5-trisphosphate production and thereby induces release of Ca(2+) from endoplasmic reticulum stores. The increased cytoplasmic Ca(2+) promotes the assembly of inflammasome components, and intracellular Ca(2+) is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1? production from CAPS patients' peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that Ca(2+) and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS.

Project description:Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [alpha-(32)P]UTP (e.g. 36.0 +/- 6.0 cpm/ng of DNA control versus 246.4 +/- 15.4 cpm/ng of DNA Ang-II, 390.1 +/- 15.5 cpm/ng of DNA L-162313 (AT1), 180.9 +/- 7.2 cpm/ng of DNA CGP42112A (AT2), p < 0.001). Ang-II application to cardiomyocyte nuclei enhanced NFkappaB mRNA expression, a response that was suppressed by co-administration of AT1R (valsartan) and/or AT2R (PD123177) blockers. Dose-response experiments with Ang-II applied to purified cardiomyocyte nuclei versus intact cardiomyocytes showed greater increases in NFkappaB mRNA levels at saturating concentrations with approximately 2-fold greater affinity upon nuclear application, suggesting preferential nuclear signaling. AT1R, but not AT2R, stimulation increased [Ca(2+)] in isolated cardiomyocyte nuclei. Inositol 1,4,5-trisphosphate receptor blockade by 2-aminoethoxydiphenyl borate prevented AT1R-mediated Ca(2+) release and attenuated AT1R-mediated transcription initiation responses. We conclude that cardiomyocyte nuclear membranes possess angiotensin receptors that couple to nuclear signaling pathways and regulate transcription. Signaling within the nuclear envelope (e.g. from intracellularly synthesized Ang-II) may play a role in Ang-II-mediated changes in cardiac gene expression, with potentially important mechanistic and therapeutic implications.

Dataset Information

Calmodulin regulates Ca2+-sensing receptor-mediated Ca2+ signaling and its cell surface expression.

Publications

Calmodulin regulates Ca2+-sensing receptor-mediated Ca2+ signaling and its cell surface expression.

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