A CONSTITUTIVELY ACTIVE MUTANT BETA(2)-ADRENERGIC RECEPTOR IS CONSTITUTIVELY DESENSITIZED AND PHOSPHORYLATED
A CONSTITUTIVELY ACTIVE MUTANT BETA(2)-ADRENERGIC RECEPTOR IS CONSTITUTIVELY DESENSITIZED AND PHOSPHORYLATED
复制标题
DOI：
10.1073/pnas.91.7.2699
复制
发表时间：
1994-03-29
影响因子：
11.1
通讯作者：
LEFKOWITZ, RJ
中科院分区：
文献类型：
--
作者：
PEI, G;SAMAMA, P;LEFKOWITZ, RJ
β 2-肾上腺素能受体（β 2AR）可通过第三胞内环的突变而组成性激活。野生型受体在没有激动剂的情况下主要以非活性构象（R）存在，而突变型受体似乎自发地采用活性构象（R*）。我们现在证明，突变体β 2 AR不仅是组成型活性的，它也是组成型脱敏和下调。为了评估突变体受体是否可以组成性地接合细胞脱敏机制的已知元件，将受体纯化并重建成磷脂囊泡。这些制剂保留了组成型活性突变体受体的基本特性：激动剂非依赖性活性[刺激鸟嘌呤核苷酸结合蛋白（G（s））-GTdR]和激动剂特异性结合亲和力增加。此外，纯化的突变体受体，在激动剂的情况下，磷酸化的重组β AR特异性激酶（β ARK）的方式与激动剂占据的野生型受体。因此，突变受体的构象等同于活性构象（R*），其刺激G*（s）蛋白并与β ARK底物相同。
The beta2-adrenergic receptor (beta2AR) can be constitutively activated by mutations in the third intracellular loop. Whereas the wild-type receptor exists predominantly in an inactive conformation (R) in the absence of agonist, the mutant receptor appears to spontaneously adopt an active conformation (R*). We now demonstrate that not only is the mutant beta2AR constitutively active, it is also constitutively desensitized and down-regulated. To assess whether the mutant receptor can constitutively engage a known element of the cellular desensitization machinery, the receptor was purified and reconstituted into phospholipid vesicles. These preparations retained the essential properties of the constitutively active mutant receptor: agonist-independent activity [to stimulate guanine nucleotide-binding protein (G(s))-GTPase] and agonist-specific increase in binding affinity. Moreover, the purified mutant receptor, in the absence of agonist, was phosphorylated by recombinant betaAR-specific kinase (betaARK) in a fashion comparable to the agonist-occupied wild-type receptor. Thus, the conformation of the mutated receptor is equivalent to the active conformation (R*), which stimulates G*(s) protein and is identical to the betaARK substrate.