Guan R, Zhang Y, Jiang J, Baumann C A, Black R A, Baumann G, Frank S J
Department of Medicine, Division of Endocrinology and Metabolism, University of Alabama, Birmingham, Alabama 35294, USA.
Endocrinology. 2001 Mar;142(3):1137-47. doi: 10.1210/endo.142.3.8030.
GH signals by interacting with GH receptor (GHR). A substantial fraction of circulating GH complexes with GH-binding protein (GHBP), which corresponds to the GHR extracellular domain. GHBP is generated by 1) alternative splicing of a common GHR precursor messenger RNA to encode secreted GHBP (the source of the vast majority of GHBP in rodents); and 2) proteolysis of the cell-associated GHR with shedding of GHBP (a mechanism operative in rabbits and humans). We previously observed that phorbol ester (PMA)-induced activation of protein kinase C (PKC) causes metalloprotease-mediated GHR proteolysis and GHBP shedding in human IM-9 lymphocytes. We now demonstrate that PMA-induced hydroxamate (IC3)-inhibitable GHR proteolysis and GHBP shedding were also detected in murine 3T3-F442A and 3T3-L1 preadipocytes and in Chinese hamster ovary (CHO) cells stably expressing rabbit GHR (rbGHR), although the degree of GHBP shedding was much smaller for murine GHR than for rabbit or human GHRs. PMA-induced GHR proteolysis in 3T3-F442A, 3T3-L1, and CHO-rbGHR cells was significantly reduced by pretreatment with mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitors, suggesting involvement of the mitogen-activated protein kinase pathway in regulating this PKC-dependent effect. In contrast, GHR proteolysis promoted by N-ethylmaleimide, although inhibited by IC3, was unaffected by inhibition of either PKC or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. Thus, different pathways leading to metalloprotease-mediated receptor proteolysis are accessed by PMA vs. N-ethylmaleimide. To determine whether other, possibly more physiologically relevant, stimuli induce GHR proteolysis, we tested effects of platelet-derived growth factor (PDGF) and serum. Treatment of serum-deprived cells with PDGF (in 3T3-F442A cells) or serum (in 3T3-F442A and CHO-rbGHR cells) promoted GHR proteolysis, which was inhibited by IC3. Interestingly, PMA-, PDGF-, and serum-induced GHR proteolysis was associated with substantial decreases in GH-induced activation of Janus kinase-2, which were also prevented by IC3. These findings suggest that inducible metalloprotease-mediated GHR proteolysis constitutes an important mechanism of receptor down-regulation and modulation of GH signaling.
生长激素（GH）通过与生长激素受体（GHR）相互作用来传递信号。循环中的生长激素有很大一部分与生长激素结合蛋白（GHBP）形成复合物，GHBP相当于GHR的细胞外结构域。GHBP的产生途径有：1）普通GHR前体信使核糖核酸通过可变剪接来编码分泌型GHBP（这是啮齿动物中绝大多数GHBP的来源）；2）细胞相关的GHR发生蛋白水解并释放出GHBP（此机制在兔和人类中起作用）。我们之前观察到，佛波酯（PMA）诱导的蛋白激酶C（PKC）激活会导致人IM-9淋巴细胞中金属蛋白酶介导的GHR蛋白水解和GHBP释放。我们现在证明，在小鼠3T3-F442A和3T3-L1前脂肪细胞以及稳定表达兔GHR（rbGHR）的中国仓鼠卵巢（CHO）细胞中，也检测到了PMA诱导的、可被异羟肟酸（IC3）抑制的GHR蛋白水解和GHBP释放，不过小鼠GHR的GHBP释放程度比兔或人类GHR小得多。用丝裂原活化蛋白激酶/细胞外信号调节激酶激酶1抑制剂预处理后，3T3-F442A、3T3-L1和CHO-rbGHR细胞中PMA诱导的GHR蛋白水解显著减少，这表明丝裂原活化蛋白激酶途径参与调节这种PKC依赖性效应。相比之下，N-乙基马来酰亚胺促进的GHR蛋白水解虽然被IC3抑制，但不受PKC或丝裂原活化蛋白激酶/细胞外信号调节激酶激酶1抑制的影响。因此，PMA和N-乙基马来酰亚胺引发金属蛋白酶介导的受体蛋白水解的途径不同。为了确定其他可能更具生理相关性的刺激是否会诱导GHR蛋白水解，我们测试了血小板衍生生长因子（PDGF）和血清的作用。用PDGF（在3T3-F442A细胞中）或血清（在3T3-F442A和CHO-rbGHR细胞中）处理血清饥饿的细胞会促进GHR蛋白水解，IC3可抑制这种水解。有趣的是，PMA、PDGF和血清诱导的GHR蛋白水解与GH诱导的Janus激酶-2激活的显著降低有关，IC3也可阻止这种降低。这些发现表明，可诱导的金属蛋白酶介导的GHR蛋白水解是受体下调和调节GH信号的重要机制。
