14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling

• Published in: PloS one Vol. 10; no. 8; p. e0136702
• Main Authors: Grant, Michael P, Cavanaugh, Alice, Breitwieser, Gerda E
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 28.08.2015; Public Library of Science (PLoS)
• Subjects: 14-3-3 protein; 14-3-3 Proteins - genetics; 14-3-3 Proteins - metabolism; Arginine; Binding; Biosynthesis; Buffers; Calcium; Calcium (extracellular); Calcium (intracellular); Calcium - metabolism; Calcium buffering; Calcium ions; Calcium Signaling - physiology; Calcium signalling; Calcium-sensing receptors; Cell Membrane - genetics; Cell Membrane - metabolism; Cell receptors; Cellular proteins; Cellular signal transduction; Defective mutant; Endoplasmic reticulum; Epilepsy; Fluorescence; Fluorescence microscopy; Genetic aspects; Glycosylation; Glycosylation - drug effects; Health aspects; HEK293 Cells; Homeostasis; Humans; Hypercalcemia; Hypocalcemia; Intracellular; Intracellular signalling; Mutation; Pancreatitis; Phosphorylation; Properties; Protein Binding; Protein Structure, Tertiary; Protein transport; Proteins; Receptors; Receptors, Calcium-Sensing - genetics; Receptors, Calcium-Sensing - metabolism; Time dependence; Tunicamycin; Tunicamycin - pharmacology; Western blotting; United States > US; Pennsylvania
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0136702

Calcium sensing receptors (CaSR) interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.