Markovic Danijela, Punn Anu, Lehnert Hendrik, Grammatopoulos Dimitris K
Endocrinology and Metabolism, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom.
Mol Endocrinol. 2008 Mar;22(3):689-706. doi: 10.1210/me.2007-0136. Epub 2007 Nov 29.
Many important physiological roles of the urocortin (UCN) family of peptides as well as CRH involve the type 2 CRH receptor (CRH-R2) and downstream activation of multiple pathways. To characterize molecular determinants of CRH-R2 functional activity, we used HEK293 cells overexpressing recombinant CRH-R2beta and investigated mechanisms involved in attenuation of CRH-R2 signaling activity and uncoupling from intracellular effectors. CRH-R2beta-mediated adenylyl cyclase activation was sensitive to homologous desensitization induced by pretreatment with either UCN-II or the weaker agonist CRH. CRH-R2beta activation induced transient beta-arrestin1 and beta-arrestin2, as well as clathrin, recruitment to the plasma membrane. Beta-arrestin2 appeared to be the main beta-arrestin subtype associated with the receptor. This was followed by CRH-R2beta endocytosis in a mechanism that exhibited distinct agonist-dependent temporal characteristics. CRH-R2beta also induced transient activation of the ERK1/2 and p38MAPK signaling cascades that peaked at 5 min and returned to basal within 20-30 min. Unlike p38MAPK, activated ERK1/2 was localized both in the cytoplasm and nucleus. Experiments employing inhibitors of receptor endocytosis showed that CRH-R2beta-MAPK interaction does not require beta-arrestin, clathrin, or receptor endocytosis. Site-directed mutagenesis studies on CRH-R2beta C terminus showed that the amino acid cassette TAAV at the end of the C terminus is important for CRH-R2beta signaling because loss of a potential phospho-acceptor site in mutant receptors containing deletion or Ala substitution of the cassette TAAV resulted in reduced ERK1/2 activation and accelerated receptor internalization. These findings provide new insights about the signaling mechanisms regulating CRH-R2beta functional activity and determining its biological responses.
促肾上腺皮质激素释放激素（CRH）家族肽类以及CRH的许多重要生理作用都涉及2型CRH受体（CRH-R2）和多条途径的下游激活。为了表征CRH-R2功能活性的分子决定因素，我们使用过表达重组CRH-R2β的HEK293细胞，并研究了参与CRH-R2信号活性减弱以及与细胞内效应器解偶联的机制。CRH-R2β介导的腺苷酸环化酶激活对用UCN-II或较弱激动剂CRH预处理诱导的同源脱敏敏感。CRH-R2β激活诱导瞬时β-抑制蛋白1和β-抑制蛋白2以及网格蛋白募集到质膜。β-抑制蛋白2似乎是与该受体相关的主要β-抑制蛋白亚型。随后是CRH-R2β以内吞作用，该机制表现出不同的激动剂依赖性时间特征。CRH-R2β还诱导ERK1/2和p38MAPK信号级联的瞬时激活，在5分钟时达到峰值，并在20-30分钟内恢复到基础水平。与p38MAPK不同，活化的ERK1/2定位于细胞质和细胞核中。采用受体内吞作用抑制剂的实验表明，CRH-R2β-MAPK相互作用不需要β-抑制蛋白、网格蛋白或受体内吞作用。对CRH-R2β C末端的定点诱变研究表明，C末端末端的氨基酸盒TAAV对CRH-R2β信号传导很重要，因为在含有盒TAAV缺失或丙氨酸替代的突变受体中潜在磷酸化接受位点的缺失导致ERK1/2激活减少和受体内化加速。这些发现为调节CRH-R2β功能活性和决定其生物学反应的信号传导机制提供了新的见解。
