Hull K L, Janssens W C, Baumbach W R, Harvey S
Department of Physiology, University of Alberta, Edmonton, Canada.
Growth Regul. 1996 Sep;6(3):165-75.
The presence of growth hormone (GH) receptor (GHR) gene transcripts and GH-binding sites in guinea pig liver suggests normal expression and translation of a GHR gene in these animals. Guinea pigs are, however, resistant to GH action and appear to lack the circulating GH-binding proteins (GHBPs) that result from alternate splicing of the GHR message or from cleavage of the extracellular binding domain of membrane GHRs. The paradoxical absence of circulating GHBPs in guinea pigs was therefore examined. The presence of GHR/GHBP mRNA in guinea pig liver was confirmed by Northern blotting. In addition to a 4.4 kb transcript that probably encodes a full-length receptor, an additional 1.9 kb transcript was detected that may encode a binding protein, although this transcript is larger than rat GHBP mRNA. The possibility that these transcripts may be translated into GHBPs was assessed immunologically. A 46 kDa protein, identical in size to rat GHBP, was specifically detected in guinea pig liver by a monoclonal antibody (MAb 4.3) raised against the hydrophilic tail of rat GHBP. A single protein of approximately 48 kDa was also detected by MAb 4.3 in proteins precipitated from guinea pig serum by a polyclonal antibody raised against the rat GHBP. This protein was slightly larger than the two proteins (46 kDa and 40 kDa) in rat serum labelled by the same method. The presence of a putative GHBP in guinea pig serum was also supported by the cross-reactivity of guinea pig serum with a monoclonal antibody (MAb 263) raised against rat GHBP. The binding of radioiodinated hGHBP to this antibody was inhibited, in a dose-related way and parallel to that of the standard, by serial dilutions of guinea pig serum, indicating immunoreactive GHBP concentrations > 500 ng/ml. Immunoreactive GHBP concentrations in other mammalian serum (from rats, rabbits, pigs, cattle, horses, goats, dogs and humans) were, in contrast, < 30 ng/ml. Guinea pig sera similarly cross-reacted, but to a lesser degree, in other radioimmunoassays for rGHBP, in which p(Ab)1 or MAb 4.3 were used as the primary antibodies. Nevertheless, despite these immunological findings, hGH binding activity could not be detected in guinea pig serum using a number of different radioligand binding assays. These results suggest the novel presence of abundant, but possibly defective, GHBP-like proteins in guinea pig serum. The immunological detection of the hydrophilic sequence of rat GHBP in guinea pig hepatic and serum proteins also suggests that GHBPs in this species arise from the truncated GHR gene transcript identified in guinea pig liver.
豚鼠肝脏中生长激素（GH）受体（GHR）基因转录本和GH结合位点的存在表明这些动物中GHR基因有正常表达和翻译。然而，豚鼠对GH作用有抗性，且似乎缺乏由GHR信息的可变剪接或膜GHRs细胞外结合域的裂解产生的循环GH结合蛋白（GHBPs）。因此，对豚鼠中循环GHBPs的矛盾缺失进行了研究。通过Northern印迹法证实了豚鼠肝脏中存在GHR/GHBP mRNA。除了一个可能编码全长受体的4.4 kb转录本外，还检测到一个额外的1.9 kb转录本，其可能编码一种结合蛋白，尽管该转录本比大鼠GHBP mRNA大。通过免疫方法评估了这些转录本是否可能翻译成GHBPs。一种大小与大鼠GHBP相同的46 kDa蛋白，在豚鼠肝脏中被针对大鼠GHBP亲水尾部产生的单克隆抗体（MAb 4.3）特异性检测到。在由针对大鼠GHBP产生的多克隆抗体从豚鼠血清中沉淀的蛋白中，MAb 4.3也检测到一种约48 kDa的单一蛋白。该蛋白比用相同方法标记的大鼠血清中的两种蛋白（46 kDa和40 kDa）略大。豚鼠血清与针对大鼠GHBP产生的单克隆抗体（MAb 263）的交叉反应性也支持了豚鼠血清中存在假定的GHBP。豚鼠血清的系列稀释以剂量相关的方式抑制了放射性碘标记的hGHBP与该抗体的结合，且与标准品平行，表明免疫反应性GHBP浓度>500 ng/ml。相比之下，其他哺乳动物血清（来自大鼠、兔子、猪、牛、马、山羊狗和人类）中的免疫反应性GHBP浓度<30 ng/ml。在其他以p(Ab)1或MAb 4.3作为一抗的rGHBP放射免疫分析中，豚鼠血清也有类似的交叉反应，但程度较轻。然而，尽管有这些免疫学发现，使用多种不同的放射性配体结合分析在豚鼠血清中均未检测到hGH结合活性。这些结果表明豚鼠血清中存在大量但可能有缺陷的GHBP样蛋白这一新颖现象。在豚鼠肝脏和血清蛋白中对大鼠GHBP亲水序列的免疫检测也表明该物种中的GHBPs源自豚鼠肝脏中鉴定出的截短GHR基因转录本。
