title: Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations.
abstract: The Ca(2+)-sensing receptor (CaSR) regulates Ca(2+) homeostasis in the body by monitoring extracellular levels of Ca(2+) ([Ca(2+)]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca(2+)]o-induced [Ca(2+)]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca(2+)]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca(2+)]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu(173), which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro(221) and Leu(173) are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia, United States of America; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, United States of America.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia, United States of America.
authors.forename: Nagaraju
authors.initials: N
authors.lastname: Mulpuri
authors.affiliation: Academic Endocrine Unit, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Fadil M
authors.initials: FM
authors.lastname: Hannan
authors.affiliation: Academic Endocrine Unit, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, United Kingdom.
authors.forename: M Andrew
authors.initials: MA
authors.lastname: Nesbit
authors.affiliation: Academic Endocrine Unit, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Rajesh V
authors.initials: RV
authors.lastname: Thakker
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia, United States of America; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, United States of America.
authors.forename: Donald
authors.initials: D
authors.lastname: Hamelberg
authors.affiliation: Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Brown
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia, United States of America; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, United States of America.
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang
mesh_terms: D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006934:Hypercalcemia / Q000235:genetics; D006996:Hypocalcemia / Q000235:genetics; D007295:Inositol Phosphates / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D010649:Phenylalanine / Q000378:metabolism* / Q000494:pharmacology; D025341:Principal Component Analysis; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D007295:Inositol Phosphates; D044169:Receptors, Calcium-Sensing; D010649:Phenylalanine; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium