Chang W, Pratt S, Chen T H, Bourguignon L, Shoback D
Endocrine Research Unit, Department of Veterans Affairs Medical Center, University of California, San Francisco, California 94121, USA.
J Biol Chem. 2001 Nov 23;276(47):44129-36. doi: 10.1074/jbc.M104834200. Epub 2001 Sep 4.
The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca(2+)] (Ca(2+)) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising Ca(2+) increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high Ca(2+) (up to 30 mm) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser(866) and Val(895) using tandem-Ala and single-site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high Ca(2+)-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha-helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.
钙受体（CaR）的C末端尾巴调节受体对配体的亲和力、脱敏作用及膜定位。为了确定牛甲状旁腺CaR中特定氨基酸在介导信号转导和细胞表面表达中的作用，我们将截短和突变的CaR cDNA转染到HEK - 293细胞中。测定了高细胞外[Ca(2+)]（Ca(2+)）增加总肌醇磷酸（InsP）生成的能力，这是磷脂酶C（PLC）激活的一个指标。通过免疫印迹和免疫细胞化学评估受体表达。在瞬时或稳定表达C末端尾巴在第895位残基（CaR-(1 - 895)）或929位残基（CaR-(1 - 929)）后被截短的受体的细胞中，提高Ca(2+)可使InsP增加到与表达野生型CaR的细胞相当的水平。在表达仅含3个残基的C末端尾巴的CaR（CaR-(1 - 866)）的细胞中，即使这些受体在膜上表达，对高Ca(2+)（高达30 mM）也没有PLC反应。我们使用串联丙氨酸和单点诱变扫描了Ser(866)和Val(895)之间的残基。两个点突变体（His(880) --> Ala和Phe(882) --> Ala CaR）在高Ca(2+)诱导的InsP生成中显示出50 - 70%的降低。这些突变体的表达水平和糖基化与野生型CaR相当，但两种受体都被严重滞留在细胞内细胞器中，并与内质网标记物BiP共定位。这表明这些受体的信号缺陷可能是由于受体向细胞表面的转运缺陷所致。CaR C末端结构域的建模表明，His(880)和Phe(882)位于C末端尾巴中第877和891位残基之间一个由15个氨基酸组成的假定α螺旋结构中。我们的研究支持这样一种观点，即C末端尾巴中的特定氨基酸以及可能独特的二级结构是CaR有效靶向到细胞表面以实现PLC激活所必需的。
