Markovic Danijela, Papadopoulou Nikolleta, Teli Thalia, Randeva Harpal, Levine Michael A, Hillhouse Edward W, Grammatopoulos Dimitris K
Endocrinology and Metabolism, Division of Clinical Sciences, Warwick Medical School, University of Warwick, Coventry, UK.
J Pharmacol Exp Ther. 2006 Dec;319(3):1032-42. doi: 10.1124/jpet.106.107441. Epub 2006 Sep 6.
Corticotropin-releasing hormone (CRH) regulates diverse biological functions in mammals, through activation of two types of specific G protein-coupled receptors that are expressed as multiple mRNA spliced variants. In most cells, the type 1alpha CRH receptor (CRH-R1alpha) preferentially activates the G(s)-adenylyl cyclase signaling cascade. CRH-R1alpha-mediated signaling activity is impaired by insertion of 29 amino acids in the first intracellular loop, a sequence modification that is characteristic of the human-specific CRH-R1beta variant. In various tissues, CRH signaling events are regulated by protein kinase C (PKC). The CRH receptors contain multiple putative PKC phosphorylation sites that represent potential targets. To investigate this, we expressed recombinant CRH-R1alpha or CRH-R1beta in human embryonic kidney 293 cells and analyzed signaling events after PKC activation. Agonist (oxytocin) or phorbol 12-myristate 13-acetate-induced activation of PKC led to phosphorylation of both CRH-R1 variants. However, CRH-R1alpha and CRH-R1beta exhibited different functional responses to PKC-induced phosphorylation, with only the CRH-R1beta susceptible to cAMP signaling desensitization. This was associated with a significant decrease of accessible CRH-R1beta receptors expressed on the cell surface. Both CRH-R1 variants were susceptible to homologous desensitization and internalization following treatment with CRH; however, PKC activation increased internalization of CRH-R1beta but not CRH-R1alpha in a beta-arrestin-independent manner. Our findings indicate that CRH-R1alpha and -R1beta exhibit differential responses to PKC-induced phosphorylation, and this might represent an important mechanism for functional regulation of CRH signaling in target cells.
促肾上腺皮质激素释放激素（CRH）通过激活两种特定的G蛋白偶联受体来调节哺乳动物的多种生物学功能，这两种受体以多种mRNA剪接变体的形式表达。在大多数细胞中，1α型CRH受体（CRH-R1α）优先激活G(s)-腺苷酸环化酶信号级联反应。CRH-R1α介导的信号活性因在第一个细胞内环中插入29个氨基酸而受损，这种序列修饰是人类特异性CRH-R1β变体的特征。在各种组织中，CRH信号事件受蛋白激酶C（PKC）调节。CRH受体含有多个假定的PKC磷酸化位点，这些位点是潜在的靶点。为了研究这一点，我们在人胚肾293细胞中表达重组CRH-R1α或CRH-R1β，并分析PKC激活后的信号事件。激动剂（催产素）或佛波酯12-肉豆蔻酸酯13-乙酸酯诱导的PKC激活导致两种CRH-R1变体的磷酸化。然而，CRH-R1α和CRH-R1β对PKC诱导的磷酸化表现出不同的功能反应，只有CRH-R1β对cAMP信号脱敏敏感。这与细胞表面表达的可及性CRH-R1β受体的显著减少有关。在用CRH处理后，两种CRH-R1变体都易发生同源脱敏和内化；然而，PKC激活以不依赖β-抑制蛋白的方式增加了CRH-R1β的内化，但没有增加CRH-R1α的内化。我们的研究结果表明，CRH-R1α和-R1β对PKC诱导的磷酸化表现出不同的反应，这可能代表了靶细胞中CRH信号功能调节的重要机制。
