Schubert R L, Puett D
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
J Mol Endocrinol. 2003 Aug;31(1):157-68. doi: 10.1677/jme.0.0310157.
Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.
人绒毛膜促性腺激素（hCG）是糖蛋白激素家族的一员，其包含一个共同的α亚基和赋予激素特异性的不同β亚基。hCG与人促黄体生成素受体（hLHR）相对较大的胞外结构域结合，hLHR是G蛋白偶联受体超家族的成员，导致细胞内cAMP产量增加。利用蛋白质工程技术，已将两个小型化的hCGβ版本分别融合到α亚基的N端，得到N-des[1-91]hCGβ-α-C和N-des[1-91,110-114]hCGβ-α-C，即hCGβ决定簇环（扩展至包括完整的安全带和羧基末端肽）与α亚基偶联的融合蛋白。在瞬时转染到HEK 293细胞并随后进行cAMP测量后，在两个系统中评估了这些单链促性腺激素类似物的生物活性。在一个系统中，每个小型化的β-α cDNA与hLHR的cDNA融合并转染到细胞中，以产生轭合的小型化hCG-hLHR复合物；在另一个系统中，每个单链小型化的β-α cDNA与hLHR的cDNA共转染，以产生非共价的小型化hCG-hLHR复合物。分别使用轭合的hCG-hLHR和hLHR作为阳性和阴性对照，使用抗体和配体结合试验确认了每个小型化hCG-hLHR复合物的表达。这两种小型化hCG导致hLHR的激活最小，表明小型化β-α融合蛋白的内在活性较弱。这些结果表明，除了决定簇环/安全带外，强效激动剂和拮抗剂还需要hCGβ的其他部分存在。
