Strader C D, Sigal I S, Register R B, Candelore M R, Rands E, Dixon R A
Proc Natl Acad Sci U S A. 1987 Jul;84(13):4384-8. doi: 10.1073/pnas.84.13.4384.
The functional significance of conserved polar amino acids within the putative transmembrane region of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster gene encoding beta AR and expression of the mutant genes in COS-7 cells. Although a substitution of aspartate at position 113 with an asparagine residue did not affect expression or processing of the protein, the resulting mutant beta AR did not show detectable binding toward the antagonist iodocyanopindolol. Replacement of the aspartate and asparagine residues at positions 79 and 318, respectively, had no effect on the affinity of the receptor toward antagonists but reduced the affinity of the receptor toward agonists by 1 order of magnitude. Furthermore, we observed that substitution of the proline at position 323 with a serine residue resulted in improper or incomplete processing of the beta AR, presumably reflecting a role for this residue in the folding of the receptor. Together with our previous results from deletion mutagenesis studies, these observations indicate that the ligand binding site involves the transmembrane region of the beta AR.
通过对编码β - 肾上腺素能受体（βAR）的仓鼠基因进行寡核苷酸定向诱变，并在COS - 7细胞中表达突变基因，研究了β - 肾上腺素能受体假定跨膜区域内保守极性氨基酸的功能意义。虽然将113位的天冬氨酸替换为天冬酰胺残基不影响蛋白质的表达或加工，但所得的突变型βAR对拮抗剂碘氰吲哚洛尔未表现出可检测到的结合。分别替换79位和318位的天冬氨酸和天冬酰胺残基对受体与拮抗剂的亲和力没有影响，但使受体与激动剂的亲和力降低了1个数量级。此外，我们观察到将323位的脯氨酸替换为丝氨酸残基导致βAR加工不当或不完全，这可能反映了该残基在受体折叠中的作用。结合我们之前缺失诱变研究的结果，这些观察结果表明配体结合位点涉及βAR的跨膜区域。
