Calmodulin interacts with the V2 vasopressin receptor: elimination of binding to the C terminus also eliminates arginine vasopressin-stimulated elevation of intracellular calcium.

Nickols HH, Shah VN, Chazin WJ, Limbird LE
J Biol Chem. 2004 279 (45): 46969-80

PMID: 15319442 · DOI:10.1074/jbc.M407351200

To identify molecules that might contribute to V2 vasopressin receptor (V2R) trafficking or signaling, we searched for novel interacting proteins with this receptor. Preliminary data, using the V2R C terminus as bait in a yeast two-hybrid screen, revealed calmodulin as a binding partner. Because calmodulin interacts with other G protein-coupled receptors, we explored this interaction and its possible functional relevance in greater detail. A Ca2+ -dependent interaction occurs between calmodulin-linked agarose and the holo-V2R as well as the V2R C terminus. Truncation and site-directed mutagenesis of the V2R C terminus revealed an involvement of an RGR sequence in this interaction. NMR studies showed that a peptide fragment of the V2R C terminus containing the RGR sequence binds to calmodulin in a Ca2+ -dependent manner with a Kd < or =1.5 microm; concentration-dependent binding of the V2R C terminus to calmodulin-agarose was used to estimate a Kd value of approximately 200 nm for this entire C-terminal sequence as expressed in mammalian cells. Madin-Darby canine kidney II cells stably expressing either wild type or a mutant V2R, in which the RGR C-terminal sequence was mutated to alanines (AAA V2R), revealed that the steady-state localization and agonist-induced internalization of the AAA V2R resembled that of the wild type V2R in polarized Madin-Darby canine kidney II cells. V2R binding of agonist similarly was unchanged in the AAA V2R, as was the concentration response for arginine vasopressin (AVP)-stimulated cAMP accumulation. Most interestingly, AVP-induced increases in intracellular Ca2+ observed for the wild type V2R were virtually eliminated for the AAA V2R. Taken together, the data suggest that a C-terminal region of the V2R important for calmodulin interaction is also important in modulation of V2R elevation of intracellular Ca2+, a prerequisite for AVP-induced fusion of aquaporin-containing vesicles with the apical surface of renal epithelial cells.

MeSH Terms (33)

Amino Acid Sequence Animals Binding, Competitive Calcium Calmodulin Cell Line Cell Membrane COS Cells Cyclic AMP Detergents DNA, Complementary Dogs Dose-Response Relationship, Drug Endocytosis Enzyme-Linked Immunosorbent Assay Glutathione Transferase Humans Hydrogen-Ion Concentration Kinetics Magnetic Resonance Spectroscopy Molecular Sequence Data Peptides Protein Binding Protein Conformation Protein Structure, Tertiary Receptors, Vasopressin Sepharose Sequence Homology, Amino Acid Signal Transduction Time Factors Transfection Two-Hybrid System Techniques Vasopressins

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