A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+.
Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.
Hu
Jianxin
J
Molecular Pathophysiology Section, NIDCD, National Institutes of Health, Bethesda, Maryland 20892, USA. jianxinh@intra.niddk.nih.gov
McLarnon
Stuart J
SJ
Mora
Stefano
S
Jiang
Jiankang
J
Thomas
Craig
C
Jacobson
Kenneth A
KA
Spiegel
Allen M
AM
D000409:Alanine / Q000737:chemistry; D000495:Allosteric Site; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007477:Ions; D007532:Isoleucine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010716:Phosphatidylinositols / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing / Q000737:chemistry*; D014162:Transfection
D007477:Ions; D010716:Phosphatidylinositols; D044169:Receptors, Calcium-Sensing; D007532:Isoleucine; D005973:Glutamine; D011392:Proline; D000409:Alanine; D002118:Calcium