The Calcium-sensing Receptor Is Localized in Caveolin-rich Plasma Membrane Domains of Bovine Parathyroid Cells

Olga Kifor,Ruben Diaz,Robert Butters,Imre Kifor,Edward M Brown

doi:10.1074/jbc.273.34.21708

Olga Kifor, Ruben Diaz + Show 3 more

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https://doi.org/10.1074/jbc.273.34.21708

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Abstract

Parathyroid cells have an intracellular machinery for parathyroid hormone (PTH) secretion that is inversely regulated by the extracellular calcium concentration (Ca2+o). The recently characterized Ca2+o-sensing receptor (CaR) is a G protein-coupled, seven-transmembrane receptor mediating the inhibitory effects of high Ca2+o on PTH secretion. The CaR's precise cell surface localization and the signal transduction pathway(s) mediating its inhibitory effects on PTH secretion have not been characterized fully. Here, we demonstrate that the CaR resides within caveolin-rich membrane domains in bovine parathyroid cells. Chief cells within bovine parathyroid glands exhibit a similar pattern of staining for caveolin-1 and for alkaline phosphatase, a glucosylphosphatidylinositol-anchored protein often enriched in caveolae. Purified caveolin-enriched membrane fractions (CEMF) from bovine parathyroid cells are highly enriched in the CaR and alkaline phosphatase. Other signaling proteins, including Gq/11, eNOS, and several protein kinase C isoforms (i.e. alpha, delta, and zeta), are also present in CEMF. Activation of the CaR by high Ca2+o increases tyrosine phosphorylation of caveolin-1 in CEMF, suggesting that CaR-mediated signal transduction potentially involved in Ca2+o-regulated processes in parathyroid cells occur in caveolae-like domains.

Highlights

Calcium ions are essential for numerous biological functions, including both vital extracellular processes as well as intracellular processes [1]
Alkaline Phosphatase and Caveolin Are Located on the Parathyroid Cell Surface—Frozen sections of bovine parathyroid glands stained for alkaline phosphatase activity (Fig. 1, A and C) show abundant punctate deposition of the insoluble reaction product solely on the surface of parathyroid chief cells, with little or no AP activity on endothelial cells (Fig. 1A)
Ca2ϩo-sensing receptor (CaR) Associates with caveolin-enriched membrane fractions (CEMF)—To determine whether the CaR is present in CEMF, bovine parathyroid glands were homogenized and fractionated using the non-detergent-based protocol of Smart et al [26], which separates caveolin-rich membrane domains from the bulk of cellular membranes and cytosolic proteins

Summary

Introduction

Calcium ions are essential for numerous biological functions, including both vital extracellular processes (i.e. blood clotting, intercellular adhesion, and skeletal integrity) as well as intracellular processes (e.g. regulation of hormonal secretion, cell division, and motility) [1]. The CaR is a G protein-coupled, seven-transmembrane receptor that is a key mediator of direct effects of Ca2ϩo on kidney and several other cells (4 –7). It plays an important role in normal calcium homeostasis and in abnormal states of mineral ion metabolism (8 –11). (iv) The CaR agonist, R-568, effectively inhibits PTH secretion and reduces Ca2ϩo in hyperparathyroid patients [12]. (v) The loss of high Ca2ϩo-evoked inhibition of PTH secretion in cultured bovine parathyroid cells correlates closely with the concomitant decreases in CaR mRNA and protein expression [13].

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