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

• Published in: The Journal of biological chemistry 2004-11, Vol.279 (45), p.46969-46980
• Main Authors: Nickols, Hilary Highfield, Shah, Vikas N, Chazin, Walter J, Limbird, Lee E
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding, Competitive; Calcium - chemistry; Calcium - metabolism; Calmodulin - chemistry; Calmodulin - metabolism; Cell Line; Cell Membrane - metabolism; COS Cells; Cyclic AMP - metabolism; Detergents - pharmacology; DNA, Complementary - metabolism; Dogs; Dose-Response Relationship, Drug; Endocytosis; Enzyme-Linked Immunosorbent Assay; Glutathione Transferase - metabolism; Humans; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Peptides - chemistry; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Receptors, Vasopressin - chemistry; Sepharose - chemistry; Sequence Homology, Amino Acid; Signal Transduction; Time Factors; Transfection; Two-Hybrid System Techniques; Vasopressins - chemistry
• ISSN: 0021-9258; 1083-351X
• 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.