Namkung Yoon, Dipace Concetta, Urizar Eneko, Javitch Jonathan A, Sibley David R
Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-9405, USA.
J Biol Chem. 2009 Dec 4;284(49):34103-15. doi: 10.1074/jbc.M109.055707. Epub 2009 Oct 8.
We investigated the regulatory effects of GRK2 on D(2) dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D(2) receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D(2) receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D(2) receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the G betagamma binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.
我们研究了GRK2对D(2)多巴胺受体信号传导的调节作用，发现该激酶以磷酸化非依赖的方式抑制受体表达和功能信号传导，显然是通过不同机制。发现GRK2的过表达会抑制细胞表面的受体表达并增强激动剂诱导的内化作用，而内源性GRK2的短发夹RNA敲低则导致细胞表面受体表达增加并减少激动剂介导的内吞作用。这些效应并非由于GRK2介导的D(2)受体磷酸化，因为磷酸化缺失的受体突变体受到类似的调节，并且GRK2催化失活突变体的过表达产生相同的效应。受体表达的抑制与GRK2与受体复合物的组成性结合相关，因为我们发现GRK2及其几个突变体能够与D(2)受体进行共免疫沉淀。激动剂预处理并未增强GRK2与受体共免疫沉淀的能力。我们还发现GRK2的过表达减弱了D(2)受体的功能偶联，并且这种活性需要GRK2的激酶活性，但不涉及受体磷酸化，因此提示存在另外的GRK2底物。有趣的是，我们发现功能信号传导的抑制也需要GRK2的Gβγ结合活性，但不涉及GRK2的N端RH结构域。我们的结果提示了一种新机制，通过该机制GRK2以独立于受体磷酸化的方式对G蛋白偶联受体信号传导进行负调节。
