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Identification and dissection of Ca(2+)-binding sites in the extracellular domain of Ca(2+)-sensing receptor.

ABSTRACT: Ca(2+)-sensing receptors (CaSRs) represent a class of receptors that respond to changes in the extracellular Ca(2+) concentration ([Ca(2+)](o)) and activate multiple signaling pathways. A major barrier to advancing our understanding of the role of Ca(2+) in regulating CaSRs is the lack of adequate information about their Ca(2+)-binding locations, which is largely hindered by the lack of a solved three-dimensional structure and rapid off rates due to low Ca(2+)-binding affinities. In this paper, we have reported the identification of three potential Ca(2+)-binding sites in a modeled CaSR structure using computational algorithms based on the geometric description and surface electrostatic potentials. Mutation of the predicted ligand residues in the full-length CaSR caused abnormal responses to [Ca(2+)](o), similar to those observed with naturally occurring activating or inactivating mutations of the CaR, supporting the essential role of these predicted Ca(2+)-binding sites in the sensing capability of the CaSR. In addition, to probe the intrinsic Ca(2+)-binding properties of the predicted sequences, we engineered two predicted continuous Ca(2+)-binding sequences individually into a scaffold protein provided by a non-Ca(2+)-binding protein, CD2. We report herein the estimation of the metal-binding affinities of these predicted sites in the CaSR by monitoring aromatic-sensitized Tb(3+) fluorescence energy transfer. Removing the predicted Ca(2+)-binding ligands resulted in the loss of or significantly weakened cation binding. The potential Ca(2+)-binding residues were shown to be involved in Ca(2+)/Ln(3+) binding by high resolution NMR and site-directed mutagenesis, further validating our prediction of Ca(2+)-binding sites within the extracellular domain of the CaSR.

SUBMITTER: Huang Y

PROVIDER: S-EPMC2867057 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Identification and dissection of Ca(2+)-binding sites in the extracellular domain of Ca(2+)-sensing receptor.

Huang Yun Y Zhou Yubin Y Yang Wei W Butters Robert R Lee Hsiau-Wei HW Li Shunyi S Castiblanco Adriana A Brown Edward M EM Yang Jenny J JJ

The Journal of biological chemistry 20070503 26

Ca(2+)-sensing receptors (CaSRs) represent a class of receptors that respond to changes in the extracellular Ca(2+) concentration ([Ca(2+)](o)) and activate multiple signaling pathways. A major barrier to advancing our understanding of the role of Ca(2+) in regulating CaSRs is the lack of adequate information about their Ca(2+)-binding locations, which is largely hindered by the lack of a solved three-dimensional structure and rapid off rates due to low Ca(2+)-binding affinities. In this paper, ...[more]

PMID: 17478419

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Project description:Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor's glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera.

Dataset Information

Identification and dissection of Ca(2+)-binding sites in the extracellular domain of Ca(2+)-sensing receptor.

Publications

Identification and dissection of Ca(2+)-binding sites in the extracellular domain of Ca(2+)-sensing receptor.

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