Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations.

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.

Identification of an L-phenylalanine binding site enhancing the cooperative responses of the calcium-sensing receptor to calcium.

Functional positive cooperative activation of the extracellular calcium ([Ca(2+)]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca(2+)]o or amino acids elicits intracellular Ca(2+) ([Ca(2+)]i) oscillations. Here, we report the central role of predicted Ca(2+)-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca(2+)-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca(2+)]o. Next, we identify an adjacent L-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca(2+)]o and L-Phe in eliciting CaSR-mediated [Ca(2+)]i oscillations. The heterocommunication between Ca(2+) and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca(2+)]o signaling by positively impacting multiple [Ca(2+)]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca(2+)]o and amino acids into intracellular signaling events.

Formation of linear polymers with pendant vinyl groups via inclusion complex mediated polymerization of divinyl monomers.

Ethylene glycol dimethacrylate (EGDMA) and ethylene glycol methacrylate 4-vinyl benzoate (EGMAVB) were shown to form 1:1 inclusion complexes with cyclodextrin and were characterized by instrumental techniques. Computational analysis showed that the bent conformation of the included divinyl monomer was more stable than its linear conformation. Complexation of the divinyl monomer with the first CD molecule offered substantial stabilization than with the second CD molecule. The vinyl group included in the CD cavity did not participate in polymerization. As a result, solvent soluble, linear polymers with pendant vinyl unsaturation per repeat unit were obtained. This was unequivocally established by the polymerization of a complex comprising CD and EGMAVB. The unreacted vinyl group can be polymerized in the subsequent step to yield cross-linked products.