Gardella T J, Luck M D, Fan M H, Lee C
Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
J Biol Chem. 1996 May 31;271(22):12820-5. doi: 10.1074/jbc.271.22.12820.
Polar residues within the transmembrane domains (TMs) of G protein-coupled receptors have been implicated to be important determinants of receptor function. We have identified mutations at two polar sites in the TM regions of the rat parathyroid hormone (PTH)/PTH-related peptide receptor, Arg-233 in TM 2 and Gln-451 in TM 7, that caused 17-200-fold reductions in the binding affinity of the agonist peptide PTH-(1-34) without affecting the binding affinity of the antagonist/partial agonist PTH-(3-34). When mutations at the TM 2 and TM 7 sites were combined, binding affinity for PTH-(1-34) was restored to nearly that of the wild type receptor. The double mutant receptors, however, were completely defective in signaling cAMP or inositol phosphate production in response to PTH-(1-34) agonist ligand. The results demonstrate that Arg-233 and Gln-451 have important roles in determining agonist binding affinity and transmembrane signaling. Furthermore, the finding that residues in TM 2 and TM 7 are functionally linked suggests that the TM domain topology of the PTH/PTH-related peptide receptor may resemble that of receptors in the rhodopsin/beta-adrenergic receptor family, for which structural and mutagenesis data suggest interactions between TMs 2 and 7.
G蛋白偶联受体跨膜结构域（TMs）内的极性残基被认为是受体功能的重要决定因素。我们在大鼠甲状旁腺激素（PTH）/PTH相关肽受体的TM区域的两个极性位点发现了突变，即TM2中的Arg-233和TM7中的Gln-451，这些突变导致激动剂肽PTH-（1-34）的结合亲和力降低了17-200倍，而不影响拮抗剂/部分激动剂PTH-（3-34）的结合亲和力。当TM2和TM7位点的突变组合时，对PTH-（1-34）的结合亲和力恢复到接近野生型受体的水平。然而，双突变受体在响应PTH-（1-34）激动剂配体时，在信号传导cAMP或肌醇磷酸产生方面完全缺陷。结果表明，Arg-233和Gln-451在决定激动剂结合亲和力和跨膜信号传导中具有重要作用。此外，TM2和TM7中的残基在功能上相关的发现表明，PTH/PTH相关肽受体的TM结构域拓扑可能类似于视紫红质/β-肾上腺素能受体家族中的受体，对于后者，结构和诱变数据表明TM2和TM7之间存在相互作用。
