Ferguson Gail, Watterson Kenneth R, Palmer Timothy M
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK.
Biochemistry. 2002 Dec 17;41(50):14748-61. doi: 10.1021/bi0262911.
In this study, we have characterized the differential effects on inhibitory adenosine receptor (AR) trafficking of disrupting predicted sites for palmitoylation and phosphorylation within each receptor's carboxyl terminus. While a Cys(302,305)Ala-mutated rat A(3)AR mutant internalizes significantly faster than the wild-type (WT) receptor in response to agonist exposure, analogous mutation of the human A(1)AR (Cys(309)Ala) had no effect on receptor internalization. Moreover, unlike the WT A(3)AR, the entire pool of internalized mutant A(3)AR is able to recycle back to the plasma membrane following agonist removal. These properties do not reflect utilization of an alternative trafficking pathway, as internalized WT and mutant A(3)ARs both accumulate into transferrin receptor-positive endosomal compartments. However, receptor accumulation into endosomes is dependent upon prior G-protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptor's carboxyl terminus, as replacement of the carboxyl-terminal domain of the human A(1)AR with the 14 GRK-phosphorylated amino acids of the rat A(3)AR confers rapid agonist-mediated endosomal accumulation of the resulting chimeric A(1)CT3AR. Sensitivity to GRK-mediated phosphorylation also dictates the distinct redistribution of arrestin3 observed upon agonist exposure. Thus, while the nonphosphorylated A(1)AR redistributes arrestin3 from the cytoplasm to punctate clusters at the plasma membrane, GRK-phosphorylated WT and Cys(302,305)Ala-mutated A(3)ARs, as well as the A(1)CT3AR chimera, each induce the redistribution of arrestin3 into punctate accumulations both at the plasma membrane and within the cytoplasm. Neither the human A(1)AR nor the rat A(3)AR colocalized with arrestin3 under basal or agonist-stimulated conditions. Together, these results demonstrate that inhibitory AR-mediated changes in arrestin3 distribution are subtype-specific, with specificity correlating with the sensitivity of the receptor's carboxyl-terminal domain to GRK phosphorylation. In the case of the rat A(3)AR, sensitivity to GRK-mediated internalization appears to be regulated in part by the integrity of putative palmitate attachment sites upstream of its GRK phosphoacceptor sites.
在本研究中，我们已明确了破坏各受体羧基末端预测的棕榈酰化和磷酸化位点对抑制性腺苷受体（AR）转运的不同影响。虽然在激动剂作用下，半胱氨酸（Cys）（302,305）突变为丙氨酸的大鼠A(3)AR突变体的内化速度明显快于野生型（WT）受体，但人A(1)AR的类似突变（Cys(309)Ala）对受体内化没有影响。此外，与WT A(3)AR不同，内化的突变体A(3)AR在去除激动剂后能够全部循环回到质膜。这些特性并不反映利用了另一种转运途径，因为内化的WT和突变体A(3)AR都积累在转铁蛋白受体阳性的内体区室中。然而，受体内化到内体中依赖于先前G蛋白偶联受体激酶（GRK）介导的受体羧基末端磷酸化，因为用人A(1)AR的羧基末端结构域替换大鼠A(3)AR的14个GRK磷酸化氨基酸会导致所得嵌合A(1)CT3AR在激动剂介导下快速在内体中积累。对GRK介导的磷酸化的敏感性也决定了激动剂作用后观察到的抑制蛋白3的不同重新分布。因此，虽然未磷酸化的A(1)AR将抑制蛋白3从细胞质重新分布到质膜上的点状簇中，但GRK磷酸化的WT和Cys(302,305)Ala突变的A(3)AR以及A(1)CT3AR嵌合体各自诱导抑制蛋白3在质膜和细胞质内重新分布成点状聚集物。在基础或激动剂刺激条件下，人A(1)AR和大鼠A(3)AR均未与抑制蛋白3共定位。总之，这些结果表明，抑制性AR介导的抑制蛋白3分布变化具有亚型特异性，特异性与受体羧基末端结构域对GRK磷酸化的敏感性相关。就大鼠A(3)AR而言，对GRK介导的内化的敏感性似乎部分受其GRK磷酸化位点上游假定的棕榈酸酯附着位点完整性的调节。
