Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates β-1 adrenergic receptor endocytosis through different pathways
Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates β-1 adrenergic receptor endocytosis through different pathways
复制标题
DOI：
10.1074/jbc.m305675200
复制
发表时间：
2003-09-12
影响因子：
4.8
通讯作者：
Rockman, HA
中科院分区：
文献类型：
--
作者：
Rapacciuolo, A;Suvarna, S;Rockman, HA
激动剂诱导的β-肾上腺素能受体(BetaAR)被G蛋白偶联受体激酶(GRKs)磷酸化，导致其脱敏和内化。蛋白激酶A(PKA)介导的β受体，特别是β(1)AR亚型的磷酸化是否也能触发内化目前尚不清楚。为了验证这一点，我们克隆了小鼠野生型β(1)AR(WTbeta(1)AR)，并分别创建了3个缺失的突变体：推测的PKA磷酸化位点(PKA-beta(1)AR)，推测的GRK磷酸化位点(GRK(-)beta(1)AR)，以及两组磷酸化位点(PKA(-)/GRK(-)beta(1)AR)。激动剂刺激后，PKA(-)β(1)AR和GRK(-)β(1)AR突变体均表现出类似的磷酸化和脱敏增加。与野生型相比，饱和浓度的激动剂只能诱导突变体50%的内化，这表明受体的PKA和GRK磷酸化都以相加的方式促进了受体的隔离。此外，与WTbeta(1)AR和PKA(-)beta(1)AR相比，GRK(-)beta(1)AR和PKA-/GRK(-)beta(1)AR的隔离不依赖于β-arrestin的募集。重要的是，笼状蛋白抑制剂消除了WTbeta(1)AR和PKA(-)beta(1)AR的激动剂依赖的内化，而小凹抑制剂仅阻止GRK(-)beta(1)AR突变体的内化。综上所述，这些数据表明：1)PKA介导的磷酸化可以触发激动剂诱导的β(1)AR内化；2)β(1)AR内化所选择的途径主要由使受体磷酸化的激酶决定，即PKA介导的磷酸化通过小凹途径引导内化，而GRK介导的磷酸化通过网状蛋白包裹的小凹引导内化。
Agonist-induced phosphorylation of beta-adrenergic receptors (betaARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of betaARs, particularly the beta(1)AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta(1)AR (WTbeta(1)AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA- beta(1)AR), the putative GRK phosphorylation sites (GRK(-)beta(1)AR), and both sets of phosphorylation sites (PKA(-)/ GRK(-)beta(1)AR). Following agonist stimulation, both PKA(-)beta(1)AR and GRK(-)beta(1)AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WTbeta(1)AR and PKA(-)beta(1)AR, sequestration of the GRK(-)beta(1)AR and PKA-/GRK(-)beta(1)AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WTbeta(1)AR and PKA(-)beta(1)AR, whereas caveolae inhibitors prevented internalization only of the GRK(-)beta(1)AR mutant. Taken together, these data demonstrate that: 1) PKA- mediated phosphorylation can trigger agonist-induced internalization of the beta(1)AR and 2) the pathway selected for beta(1)AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.