Tchelet A, Staten N R, Creely D P, Krivi G G, Gertler A
Department of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
J Endocrinol. 1995 Mar;144(3):393-403. doi: 10.1677/joe.0.1440393.
The cDNA of the extracellular domain of bovine prolactin receptor (bPRLR-ECD) was cloned and expressed at high yield as an insoluble protein in Escherichia coli. This protein was solubilized, refolded and purified to > 98% homogeneity yielding 80 mg of monomeric fraction per 2 litres of induced culture. Its molecular mass was 25.7 kDa, as determined by SDS-PAGE in the absence of reducing agent and 24 kDa by gel filtration on a Superdex column. Binding experiments revealed that bPRLR-ECD binds to human (h) GH (hGH) with high affinity, whereas its affinity for ovine (o) or bovine (b) prolactins (PRLs) was lower and for bovine placental lactogen (bPL) very low. The affinity of bPRLR-ECD for the latter three hormones was, however, much higher than that of membrane-embedded or solubilized bPRLR. The stoichiometries of interaction of bPRLR-ECD with hGH, oPRL, bPRL and bPL were determined by gel-filtration chromatography. Even at a 3:1 ECD excess, only 1:1 complexes were detected at microM concentrations of ECD and ligand. At an up to 32-fold dilution, the complexes with oPRL, bPRL, and particularly bPL, underwent progressive dissociation, whereas the complex with hGH remained stable. Although all four hormones exhibited nearly identical activity in the Nb2 lymphoma cell bioassay, the ability of bPRLR-ECD to inhibit hormonal mitogenic activities differed, generally reflecting its affinity for the respective hormones. In view of these and previous results, we suggest that, unlike in the GH:GHR-ECD interaction, neither the stoichiometry of interaction of bovine or other PRLR-ECDs nor the affinity constants can predict the biological potency of the different lactogenic hormones.
牛催乳素受体胞外域（bPRLR-ECD）的cDNA被克隆，并作为不溶性蛋白在大肠杆菌中高效表达。该蛋白经溶解、复性和纯化后，纯度>98%，每2升诱导培养物可获得80毫克单体级分。在无还原剂的情况下，通过SDS-PAGE测定其分子量为25.7 kDa，在Superdex柱上进行凝胶过滤测定为24 kDa。结合实验表明，bPRLR-ECD与人生长激素（hGH）具有高亲和力，而其对绵羊（o）或牛（b）催乳素（PRL）的亲和力较低，对牛胎盘催乳素（bPL）的亲和力非常低。然而，bPRLR-ECD对后三种激素的亲和力远高于膜包埋或溶解的bPRLR。通过凝胶过滤色谱法测定了bPRLR-ECD与hGH、oPRL、bPRL和bPL相互作用的化学计量比。即使ECD过量3倍，在微摩尔浓度的ECD和配体下也仅检测到1:1复合物。在高达32倍稀释时，与oPRL、bPRL特别是bPL形成的复合物逐渐解离，而与hGH形成的复合物保持稳定。尽管所有四种激素在Nb2淋巴瘤细胞生物测定中表现出几乎相同的活性，但bPRLR-ECD抑制激素促有丝分裂活性的能力有所不同，这通常反映了其对相应激素的亲和力。鉴于这些以及之前的结果，我们认为，与GH:GHR-ECD相互作用不同，无论是牛或其他PRLR-ECD相互作用的化学计量比还是亲和常数，都无法预测不同催乳激素的生物学效力。
