Abstract
The extracellular calcium-sensing receptor (CaR) forms a disulfide-linked dimer through cysteine residues within its N-terminal extracellular domain (ECD). However, these disulfide linkages are dispensable for the formation of the dimeric CaR and for the functional reconstitution of two inactive CaRs. In this study, using molecular modeling, mutagenesis, and biochemical and biophysical analyses, we examined the importance of two leucine residues, Leu-112 and Leu-156, in the ECD of the CaR for the non-covalent dimerization and functional reconstitution. We found that the mutant receptor carrying L112S and L156S still exists mostly as a covalently linked dimer and has a significantly higher apparent affinity for calcium than the wild-type receptor. However, a combination of four mutations, L112S, L156S, C129S, and C131S, significantly reduces receptor dimerization and markedly inactivates the CaR. We also found that L112S and L156S mediate the non-covalent intermolecular interactions important for functional reconstitution. Because mutating either the two cysteines or the two leucines enhances the apparent ligand affinity of the CaR, it is likely that the changes in intermolecular relationships between two receptor protomers linked by these leucines and cysteines are essential for receptor activation. Moreover, these mutations are unlikely to have negative effects on the secondary structure of each protomer of the dimeric receptor. Thus, the detrimental effects of the combined mutations on the function of the CaR further suggest that CaR dimerization through its ECD is essential for the formation of a functional tertiary structure of the CaR.
Highlights
The extracellular calcium ([Ca2ϩ]o)1-sensing receptor (CaR) plays a key role in the sensing of [Ca2ϩ]o by homeostatic tissues such as parathyroid and kidney [1]
The interprotomer disulfide linkages of both the calcium-sensing receptor (CaR) and metabotropic glutamate receptors (mGluRs) are formed only between dimeric extracellular domains (ECDs) protomers [3, 10, 11]. These disulfide linkages are dispensable for the dimerization and the functional reconstitution of the CaR [3], a collaborative involvement of two ECD protomers of the CaR is important in the sensing of [Ca2ϩ]o [4]
Studies of the possible sites of interprotomer interactions have suggested that dimerization of G protein-coupled receptors (GPCRs) could be mediated by hydrophobic interactions, involving associations of the extracellular domains (ECDs), transmembrane domains (TMDs), and/or C-terminal tails (Ctails) [13]
Summary
Site-directed Mutagenesis—We used the site-directed mutagenesis approach described by Kunkel [15] to produce mutated receptors in which the leucine residues at positions 112 and 156 on the putative dimer interface of the human CaR were mutated individually to serines. The point mutations in the CaR that are generated by site-directed mutagenesis were moved into YFP- and CFP-fused CaR by subcloning the mutation-containing cassettes, as described previously [16]. Detection of Expressed CaR on the Cell Surface—Before whole cell lysates were prepared, intact HEK293 cells transiently transfected with FLAG-tagged CaR were labeled with 1 mM ImmunoPure N-hydroxysulfosuccinimidobiotin (Pierce), a membrane-impermeable biotinylation reagent, as described previously [2]. Where IFRET is the intensity of FRET in CFP- and YFP-co-expressed cells measured by the FRET filter set and CCFP and CYFP are the contributions from the emission of CFP and direct excitation of YFP, respectively [17]. CYFP in co-transfected cells with FRET filter IYFP in co-transfected cells with YFP filter ϭ
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