Mahan L C, Koachman A M, Insel P A
Proc Natl Acad Sci U S A. 1985 Jan;82(1):129-33. doi: 10.1073/pnas.82.1.129.
We have used wild-type and variants of the T-lymphoma cell line S49 to explore internalization and down-regulation of adenylate cyclase-linked beta-adrenergic receptors. Internalization was defined by the loss in "surface receptors" detected at 4 degrees C on intact cells by the antagonists [3H]CGP-12177 or [125I]iodocyanopindolol, whereas down-regulation was defined as the loss in total cellular content of receptors [( 125I]iodocyanopindolol binding assayed at 37 degrees C). In wild-type cells, the beta-adrenergic agonist isoproterenol induced a rapid (t 1/2, approximately equal to 1 min) and reversible loss in surface receptors. The surface sites were lost at a rate similar to the rate of desensitization of beta-adrenergic receptor-mediated cyclic AMP generation of S49 cells. A series of S49 variants (cyc-, UNC, H21a) having lesions in NS (the guanine nucleotide binding protein that couples beta-receptors to adenylate cyclase) or with absent cAMP-dependent protein kinase activity (kin-), had a loss in surface sites that was equivalent to that of wild-type cells. By contrast, S49 variant cells having lesions in NS showed variable rates and extents of down-regulation of beta-adrenergic receptors. In wild-type and kin- S49 cells, beta-receptors down-regulated with a t 1/2 of approximately equal to 4 hr. Down-regulation was blunted in the cyc- and UNC variants that have altered coupling of receptors to NS, but it was faster in the H21a variant that retains receptor-NS interaction. Recovery of receptors after down-regulation occurred at a similar rate (t 1/2, approximately equal to 6 hr) in wild-type, UNC, and H21a cells. These results demonstrate that internalization of beta-adrenergic receptors may be necessary, but is not sufficient, to explain agonist-induced receptor down-regulation in S49 cells. The variable expression in the development of down-regulation in S49 variants implies that receptor-NS interaction regulates the fate of receptors linked to the stimulation of adenylate cyclase.
我们利用T淋巴瘤细胞系S49的野生型及变体来探究腺苷酸环化酶偶联的β - 肾上腺素能受体的内化及下调过程。内化作用通过完整细胞在4℃时由拮抗剂[³H]CGP - 12177或[¹²⁵I]碘氰吲哚洛尔检测到的“表面受体”损失来定义，而下调则定义为受体总细胞含量的损失（在37℃下测定[¹²⁵I]碘氰吲哚洛尔结合）。在野生型细胞中，β - 肾上腺素能激动剂异丙肾上腺素诱导表面受体迅速（t₁/₂，约等于1分钟）且可逆性损失。表面位点的损失速率与S49细胞中β - 肾上腺素能受体介导的环磷酸腺苷生成脱敏速率相似。一系列在NS（将β受体与腺苷酸环化酶偶联的鸟嘌呤核苷酸结合蛋白）中有损伤或缺乏环磷酸腺苷依赖性蛋白激酶活性（kin -）的S49变体（cyc -、UNC、H21a），其表面位点的损失与野生型细胞相当。相比之下，在NS中有损伤的S49变体细胞显示出β - 肾上腺素能受体下调的速率和程度各不相同。在野生型和kin - S49细胞中，β受体下调的t₁/₂约等于4小时。在cyc -和UNC变体中，由于受体与NS的偶联改变，下调作用减弱，但在保留受体 - NS相互作用的H21a变体中下调更快。在野生型、UNC和H21a细胞中，下调后受体的恢复以相似的速率（t₁/₂，约等于6小时）发生。这些结果表明，β - 肾上腺素能受体的内化可能是必要条件，但不足以解释S49细胞中激动剂诱导的受体下调。S49变体下调过程中表达的差异意味着受体 - NS相互作用调节与腺苷酸环化酶刺激相关的受体命运。
