Kouhen O M, Wang G, Solberg J, Erickson L J, Law P Y, Loh H H
Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455-0217, USA.
J Biol Chem. 2000 Nov 24;275(47):36659-64. doi: 10.1074/jbc.M006788200.
Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.
用激动剂[d-青霉胺(2,5)]脑啡肽(DPDPE)处理表达δ-阿片受体的HEK293细胞，导致该受体迅速磷酸化。我们构建了受体羧基末端潜在磷酸化位点(丝氨酸/苏氨酸)的几个突变体，以确定受体磷酸化位点以及激动剂诱导的脱敏和内化。将丝氨酸和苏氨酸替换为丙氨酸，并在HEK293细胞中瞬时和稳定表达相应的突变体。我们发现只有两个残基，即苏氨酸(358)和丝氨酸(363)被磷酸化，其中丝氨酸(363)对DPDPE诱导的受体磷酸化至关重要。此外，通过丙氨酸和天冬氨酸替换，我们发现受体的磷酸化是分层的，丝氨酸(363)是主要的磷酸化位点。在此，我们证明，通过腺苷酸环化酶活性测量，DPDPE诱导的快速受体脱敏和受体内化与苏氨酸(358)和丝氨酸(363)的磷酸化密切相关，其中苏氨酸(358)参与受体内化。
