Frick G P, Tai L R, Baumbach W R, Goodman H M
Department of Physiology, University of Massachusetts Medical School, Worcester 01655-0127, USA.
Endocrinology. 1998 Jun;139(6):2824-30. doi: 10.1210/endo.139.6.6047.
Two isoforms of the GH receptor, the full-length receptor (GHRL) and a short isoform (GHRS) that lacks the transmembrane and intracellular domains of GHRL, have been analyzed in rat tissue extracts by Western blotting and immunoprecipitation. Although quantitative estimates of GHRS and GHRL based on coprecipitation of [125I]GH indicated similar amounts of both isoforms in tissue extracts, the 110 kDa band corresponding to GHRL was generally not detected on Western blots without enrichment by immunoprecipitation. Two bands with electrophoretic mobilities corresponding to 38 and 42 kDa were present in extracts prepared from liver, muscle, and adipocytes. Western blots of the GH binding protein in rat serum also revealed two bands, but these had electrophoretic mobilities corresponding to 44 and 52 kDa. After digestion by endoglycosidase F, a single band with an electrophoretic mobility corresponding to 31 kDa was detected in samples from adipocytes, liver or serum, indicating that GHRS retained in tissues is glycosylated less extensively than that in rat serum. Digestion with neuraminidase indicated that the smaller glycoproteins in tissue extracts lack sialic acid residues that are present in serum samples. Furthermore, endoglycosidase H degraded GHRS in liver extracts to a 31 kDa band but did not degrade serum samples, suggesting that tissues retain a high mannose form of GHRS. The abundance of GHRS or GHRL in tissues from male, virgin female, and pregnant rats was estimated from the amount of 125I-GH that was bound to each isoform after immunoprecipitation. Liver contained more than 10 times as much GHRS per gram of tissue as fat or muscle. In liver, muscle, and fat, the amount of GHRS exceeded that of GHRL, sometimes by as much as 6-fold. GHBP levels in serum of females exceeded those in males, and rose even higher in pregnant females. The abundance of GHRS in all tissue extracts paralleled serum levels. In muscle and fat, the levels of GHRL did not differ in male, female and pregnant rats, whereas in liver, the pattern was similar to the GHRS pattern. In all tissues, pools of GHRS exceeded those of GHRL by a factor that grew larger as tissue and serum levels increased. The half life of GHBP in serum was estimated to be 2.4 h in rats treated with cycloheximide, whereas that of GHRS was 20 min in liver and 8.5 h in fat. These results suggest that GHRS is synthesized in liver 8 times faster than it is released into serum, whereas synthesis in fat is less than 30% of the rate at which it is released into serum by all tissues. Therefore, liver appears to be the major source of GHBP in serum. Although secretion into the circulatory system accounts for little or perhaps none of its turnover in some tissues, GHRS pools in tissues do appear to be regulated, suggesting that GHRS may function primarily in the cells in which it is synthesized.
通过蛋白质免疫印迹法和免疫沉淀法，对大鼠组织提取物中的两种生长激素（GH）受体亚型进行了分析，即全长受体（GHRL）和缺少GHRL跨膜及细胞内结构域的短亚型（GHRS）。虽然基于[125I]GH共沉淀对GHRS和GHRL进行的定量估计表明，组织提取物中两种亚型的含量相似，但在未经免疫沉淀富集的蛋白质免疫印迹中，通常检测不到与GHRL相对应的110 kDa条带。从肝脏、肌肉和脂肪细胞制备的提取物中存在两条电泳迁移率分别对应于38 kDa和42 kDa的条带。大鼠血清中GH结合蛋白的蛋白质免疫印迹也显示出两条条带，但其电泳迁移率分别对应于44 kDa和52 kDa。经内切糖苷酶F消化后，在脂肪细胞、肝脏或血清样品中检测到一条电泳迁移率对应于31 kDa的单一条带，这表明组织中保留的GHRS糖基化程度低于大鼠血清中的GHRS。用神经氨酸酶消化表明，组织提取物中较小的糖蛋白缺乏血清样品中存在的唾液酸残基。此外，内切糖苷酶H将肝脏提取物中的GHRS降解为31 kDa条带，但未降解血清样品，这表明组织中保留的是高甘露糖形式的GHRS。通过免疫沉淀后与每种亚型结合的125I-GH的量，估算了雄性、未孕雌性和妊娠大鼠组织中GHRS或GHRL的丰度。每克肝脏组织中GHRS的含量比脂肪或肌肉中的含量多10倍以上。在肝脏、肌肉和脂肪中，GHRS的含量超过了GHRL，有时高达6倍。雌性血清中的GHBP水平超过雄性，在妊娠雌性中甚至更高。所有组织提取物中GHRS的丰度与血清水平平行。在肌肉和脂肪中，雄性、雌性和妊娠大鼠的GHRL水平没有差异，而在肝脏中，其模式与GHRS模式相似。在所有组织中，GHRS的总量超过GHRL的总量，且随着组织和血清水平的升高，这一倍数差变得更大。在用环己酰亚胺处理的大鼠中，血清中GHBP的半衰期估计为2.4小时，而肝脏中GHRS的半衰期为20分钟，脂肪中为8.5小时。这些结果表明，肝脏中GHRS的合成速度比释放到血清中的速度快8倍，而脂肪中的合成速度不到所有组织释放到血清中的速度的30%。因此，肝脏似乎是血清中GHBP的主要来源。尽管在某些组织中，分泌到循环系统中的量在其周转中占比很小或可能没有，但组织中的GHRS池似乎确实受到调节，这表明GHRS可能主要在其合成的细胞中发挥作用。
