Sibley D R, Strasser R H, Benovic J L, Daniel K, Lefkowitz R J
Proc Natl Acad Sci U S A. 1986 Dec;83(24):9408-12. doi: 10.1073/pnas.83.24.9408.
Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.
细胞或组织长时间暴露于药物或激素（如儿茶酚胺）会导致对该物质的进一步刺激产生不应性状态，即同源脱敏。就与腺苷酸环化酶偶联的β-肾上腺素能受体而言，这一过程已被证明与通过不依赖环磷酸腺苷（cAMP）的过程使受体从细胞表面隔离密切相关。最近，我们发现青蛙红细胞模型系统中的同源脱敏也与β-肾上腺素能受体磷酸化增加有关。我们现在提供证据表明，在激动剂诱导的同源脱敏过程中，β-肾上腺素能受体的磷酸化状态调节其与腺苷酸环化酶的功能偶联、亚细胞易位以及向细胞表面的再循环。此外，我们表明受体磷酸化可被一种与隔离的亚细胞区室特异性相关的磷酸酶逆转。在23摄氏度时，β-肾上腺素能受体磷酸化、隔离和腺苷酸环化酶脱敏的时间进程相同，无延迟发生，半衰期为30分钟，约3小时达到最大值。细胞裂解后，隔离的β-肾上腺素能受体可在轻膜囊泡部分中部分回收，该部分可通过差速离心与质膜分离。随着隔离的受体表现出与基础条件下观察到的相似的磷酸盐/受体化学计量比，隔离的囊泡部分中β-肾上腺素能受体磷酸化增加显然被逆转。高水平的β-肾上腺素能受体磷酸酶活性似乎与隔离的囊泡膜相关。通过在磷脂囊泡中用其生化效应器鸟嘌呤核苷酸调节蛋白（Ns）重组纯化的受体并评估受体刺激的Ns的GTP酶活性，检测了磷酸化β-肾上腺素能受体的功能活性。与对照相比，从脱敏细胞中纯化的磷酸化β-肾上腺素能受体在激活Ns GTP酶活性方面效果较差。这些结果表明，β-肾上腺素能受体的磷酸化导致其功能解偶联并从细胞表面物理易位到隔离的膜结构域。在隔离区室中，磷酸化被逆转，从而使受体能够再循环回到细胞表面并与腺苷酸环化酶重新偶联。
