Mahale S D, Cavanagh J, Schmidt A, MacColl R, Dias J A
Wadsworth Center, David Axelrod Institute for Public Health, New York State Department of Health, Albany, New York 12208, USA.
J Biol Chem. 2001 Apr 13;276(15):12410-9. doi: 10.1074/jbc.M100115200. Epub 2001 Jan 16.
It is generally held with respect to heterotrimeric guanine nucleotide binding protein-coupled receptors that binding of ligand stabilizes a conformation of receptor that activates adenylyl cyclase. It is not formally appreciated if, in the case of G-protein-coupled receptors with large extracellular domains (ECDs), ECDs directly participate in the activation process. The large ECD of the glycoprotein hormone receptors (GPHRs) is 350 amino acids in length, composed of seven leucine-rich repeat domains, and necessary and sufficient for high affinity binding of the glycoprotein hormones. Peptide challenge experiments to identify regions in the follicle-stimulating hormone (FSH) receptor (FSHR) ECD that could bind its cognate ligand identified only a single synthetic peptide corresponding to residues 221-252, which replicated a leucine-rich repeat domain of the FSHR ECD and which had intrinsic activity. This peptide inhibited human FSH binding to the human FSHR (hFSHR) and also inhibited human FSH-induced signal transduction in Y-1 cells expressing recombinant hFSHR. The hFSHR-(221-252) domain was not accessible to anti-peptide antibody probes, suggesting that this domain resides at an interface between the hFSHR ECD and transmembrane domains. CD spectroscopy of the peptide in dodecyl phosphocholine micelles showed an increase in the ordered structure of the peptide. CD and NMR spectroscopies of the peptide in trifluoroethanol confirmed that hFSHR-(221-252) has the propensity to form ordered secondary structure. Importantly and consistent with the foregoing results, dodecyl phosphocholine induced a significant increase in the ordered secondary structure of the purified hFSHR ECD as well. These data provide biophysical evidence of the influence of environment on GPHR ECD subdomain secondary structure and identify a specific activation domain that can autologously modify GPHR activity.
一般认为，对于异源三聚体鸟嘌呤核苷酸结合蛋白偶联受体，配体的结合会稳定受体的一种构象，从而激活腺苷酸环化酶。对于具有大细胞外结构域（ECD）的G蛋白偶联受体，ECD是否直接参与激活过程尚未得到正式认可。糖蛋白激素受体（GPHR）的大ECD长度为350个氨基酸，由七个富含亮氨酸的重复结构域组成，是糖蛋白激素高亲和力结合所必需且足够的。通过肽挑战实验来鉴定促卵泡激素（FSH）受体（FSHR）ECD中能够结合其同源配体的区域，结果仅鉴定出一个对应于残基221 - 252的合成肽，该肽重复了FSHR ECD的一个富含亮氨酸的重复结构域且具有内在活性。该肽抑制人FSH与人FSH受体（hFSHR）的结合，也抑制人FSH在表达重组hFSHR的Y - 1细胞中诱导的信号转导。hFSHR - (221 - 252)结构域不能被抗肽抗体探针识别，这表明该结构域位于hFSHR ECD和跨膜结构域之间的界面处。该肽在十二烷基磷酸胆碱胶束中的圆二色光谱显示肽的有序结构增加。该肽在三氟乙醇中的圆二色光谱和核磁共振光谱证实hFSHR - (221 - 252)具有形成有序二级结构的倾向。重要的是，与上述结果一致，十二烷基磷酸胆碱也显著增加了纯化的hFSHR ECD的有序二级结构。这些数据提供了环境对GPHR ECD亚结构域二级结构影响的生物物理证据，并鉴定出一个可以自主调节GPHR活性的特定激活结构域。
