Ozcelebi F, Miller L J
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
J Biol Chem. 1995 Feb 17;270(7):3435-41. doi: 10.1074/jbc.270.7.3435.
The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist-specific receptor regulation.
大鼠胰腺腺泡细胞上的胆囊收缩素（CCK）受体是一种G蛋白偶联受体，在同源和异源激动剂刺激下会发生磷酸化。在本研究中，我们研究了受体磷酸化的化学计量，并利用溴化氰裂解后的一维磷酸肽图谱来证明，在这些处理后，第三个细胞内环是该受体磷酸化的主要区域。在每摩尔受体平均5摩尔磷酸中，超过95%位于第三个环上，其余位于羧基末端尾巴。丝氨酸残基是超过95%磷酸化的位点，苏氨酸占其余部分，未检测到磷酸酪氨酸。此外，我们利用枯草杆菌蛋白酶裂解后的二维磷酸肽图谱来鉴定CCK受体磷酸化的不同位点，这些位点取决于用于刺激该细胞的激动剂。在用不同的蛋白激酶C激动剂刺激腺泡细胞后，观察到磷酸化位点的定性和定量差异。此外，图谱上不同的磷酸肽被鉴定为仅由天然CCK占据受体刺激的一种对星形孢菌素不敏感的激酶活性的底物，并且被认为代表G蛋白偶联受体激酶家族成员的作用位点。这代表了一种灵敏且强大的方法，适用于存在于其天然细胞环境中的稀少受体，以评估磷酸化模式可能存在的差异，这在激动剂特异性受体调节中可能很重要。
