Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

Chen Zhang,Yun Huang,Yusheng Jiang,Nagaraju Mulpuri,Ling Wei,Donald Hamelberg,Edward M Brown,Jenny J Yang

doi:10.1074/jbc.m113.537357

Chen Zhang, Yun Huang + Show 6 more

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https://doi.org/10.1074/jbc.m113.537357

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Abstract

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.

Highlights

IntroductionResults: An L-Phe binding site at the calcium-sensing receptor (CaSR) hinge region globally enhances its cooperative activation by Ca2ϩ
The calcium-sensing receptor (CaSR) is a key mediator of Ca2ϩ homeostasis in vivo
HEK293 cells were transiently transfected with the WT CaSR or Ca2ϩ binding-related CaSR mutants, and after 48 h the cells were loaded with Fura-2 as described under

Summary

Introduction

Results: An L-Phe binding site at the CaSR hinge region globally enhances its cooperative activation by Ca2ϩ. Functional positive cooperative activation of the extracellular calcium ([Ca2؉]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca2؉]o or amino acids elicits intracellular Ca2؉ ([Ca2؉]i) oscillations. We report the central role of predicted Ca2؉-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2؉-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2؉]o. We identify an adjacent L-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2؉]o and L-Phe in eliciting CaSR-mediated [Ca2؉]i oscillations. The heterocommunication between Ca2؉ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2؉]o signaling by positively impacting multiple [Ca2؉]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 [Ca2؉]o and amino acids into intracellular signaling events

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