Frick G P, Goodman H M
Department of Physiology, University of Massachusetts Medical School, Worcester 01655.
Endocrinology. 1992 Dec;131(6):3083-90. doi: 10.1210/endo.131.6.1446642.
Two mRNA transcripts that are believed to be alternately spliced products of the GH receptor gene have been reported in a variety of rat tissues. The smaller (1.2 kilobases) transcript was cloned from an adipocyte library, sequenced, and found to encode a protein identical to the soluble GH-binding protein (GHBP) in plasma. An assay that is specific for the short isoform of the GH receptor, often referred to as the GHBP, has been developed using a rabbit antiserum that recognizes the unique amino acid sequence at its carboxyl end. The assay depends upon immunoprecipitation of a complex consisting of [125I]human GH, the binding protein, antiserum, and protein-A cross-linked to agarose beads. To validate the assay, samples of rat plasma were analyzed and found to contain sufficient binding protein to bind 1.46 pmol (32 ng) GH/ml, with an affinity of 2.7 x 10(9) M-1. In adipocyte extracts, binding protein activity was sufficient to bind 61 fmol GH/g tissue, with an affinity of 2.3 x 10(9) M-1. The binding protein was found primarily in the particulate fraction of adipocytes, and it is estimated that adipocytes contain approximately 7000 copies of the binding protein/cell. Only 10% of the binding activity was present in the high speed supernatant of adipocyte homogenates, and soluble binding protein did not appear to be released into the incubation medium when adipocytes were incubated in vitro. A 50-kilodalton (kDa) 35S-labeled protein that may be a glycosylated form of the binding protein was immunoprecipitated from both the soluble and particulate fractions of adipocyte extracts by the antiserum, and addition of the synthetic peptide antigen blocked immunoprecipitation of this protein. A 150-kDa protein in the high speed supernatant fraction was also specifically immunoprecipitated by the antiserum. Although it is unlikely to be a glycosylated form of the binding protein, it may cross-react with the antiserum or perhaps be coprecipitated, because it interacts with the binding protein. In addition, 38- and 42-kDa bands were specifically immunoprecipitated from the detergent-treated particulate fraction of adipocyte extracts that were enriched for the binding protein by adsorption to immobilized GH. We conclude that 1) adipocytes synthesize the short isoform of the GH receptor, and that this protein is primarily associated with a membrane fraction of the cells; and 2) the GHBP expressed in adipocytes is not released into the incubation medium and differs in size from the GHBPs in rat plasma.(ABSTRACT TRUNCATED AT 400 WORDS)
据报道，在多种大鼠组织中存在两种信使核糖核酸转录本，它们被认为是生长激素（GH）受体基因的可变剪接产物。较小的（1.2千碱基）转录本是从脂肪细胞文库中克隆出来的，经过测序，发现其编码的蛋白质与血浆中的可溶性GH结合蛋白（GHBP）相同。利用一种兔抗血清开发了一种针对GH受体短异构体（通常称为GHBP）的检测方法，该抗血清可识别其羧基末端独特的氨基酸序列。该检测方法依赖于对由[125I]人GH、结合蛋白、抗血清以及与琼脂糖珠交联的蛋白A组成的复合物进行免疫沉淀。为验证该检测方法，对大鼠血浆样本进行了分析，发现其中含有足够的结合蛋白，可结合1.46皮摩尔（32纳克）GH/毫升，亲和力为2.7×10⁹ M⁻¹。在脂肪细胞提取物中，结合蛋白活性足以结合61飞摩尔GH/克组织，亲和力为2.3×10⁹ M⁻¹。结合蛋白主要存在于脂肪细胞的颗粒部分，据估计脂肪细胞每个细胞含有约7000个结合蛋白拷贝。在脂肪细胞匀浆的高速上清液中仅存在10%的结合活性，并且当脂肪细胞在体外培养时，可溶性结合蛋白似乎不会释放到培养液中。一种50千道尔顿（kDa）的³⁵S标记蛋白可能是结合蛋白的糖基化形式，可被抗血清从脂肪细胞提取物的可溶性和颗粒部分中免疫沉淀出来，并加入合成肽抗原可阻断该蛋白的免疫沉淀。高速上清液部分中的一种150 kDa蛋白也可被抗血清特异性免疫沉淀。尽管它不太可能是结合蛋白的糖基化形式，但它可能与抗血清发生交叉反应，或者可能是共沉淀，因为它与结合蛋白相互作用。此外，从经去污剂处理的脂肪细胞提取物颗粒部分中可特异性免疫沉淀出38 kDa和42 kDa条带，这些颗粒部分通过吸附固定化GH而富含结合蛋白。我们得出结论：1）脂肪细胞合成GH受体的短异构体，并且该蛋白主要与细胞的膜部分相关；2）脂肪细胞中表达的GHBP不会释放到培养液中，并且其大小与大鼠血浆中的GHBP不同。（摘要截短至400字）
