Qiu R, Tsao J, Kwan W K, Schimmer B P
Banting and Best Department of Medical Research, University of Toronto Ontario, Canada.
Mol Endocrinol. 1996 Dec;10(12):1708-18. doi: 10.1210/mend.10.12.8961279.
A family of mutants isolated from the Y1 mouse adrenal cell line on the basis of their resistance to the growth inhibitory effects of forskolin have an underlying mutation that affects the activity of adenylyl cyclase. As part of the mutant phenotype, adenylyl cyclase is partially resistant to activation by forskolin, completely insensitive to ACTH, and fully responsive to NaF; the levels of Gs alpha and G1 alpha in plasma membrane fractions are decreased; and the activity of G beta/gamma is impaired. In the present study, we examine the basis for the complex phenotype associated with forskolin resistance to better understand the factors that contribute to the regulation of adenylyl cyclase activity. We demonstrate that the resistance of these mutants to ACTH results from the failure to express ACTH receptor transcripts. Transfection of these mutants with a gene encoding the mouse beta 2-adrenergic receptor led to the recovery of transformants with normal receptor-G protein coupling and with increased levels of Gs alpha and G1 alpha that approached those in parental Y1 cells. These beta 2-adrenergic receptor transformants, nonetheless, remained resistant to forskolin and ACTH. Two spontaneous Y1 mutants, Y6 and OS3, previously characterized as ACTH-resistant clones that failed to accumulate ACTH receptor transcripts, were shown to be forskolin resistant and to contain less Gs alpha in membrane fractions, indicating that forskolin resistance, failure to express the ACTH receptor, and the consequent reduction in Gs alpha are closely linked. Expression of the human ACTH receptor in Y6 and OS3 cells restored ACTH-responsive adenylyl cyclase activity and increased the level of Gs alpha, but did not otherwise reverse the forskolin-resistant phenotype. Together, these results demonstrate that mutations to forskolin resistance have downstream consequences that result in the loss of ACTH receptor expression and the consequent reduction in levels of membrane-associated G alpha subunits. The results further suggest that G protein-coupled receptors may have a stabilizing influence on G alpha subunits associated with the cell membrane. According to current models, forskolin activates adenylyl cyclase by forming a ternary complex with adenylyl cyclase and Gs alpha. Our results suggest that this model may be incomplete and that an additional component, acting directly or indirectly, is required for optimal activation of adenylyl cyclase by forskolin.
从Y1小鼠肾上腺细胞系中分离出的一系列突变体，基于它们对福斯高林生长抑制作用的抗性，具有影响腺苷酸环化酶活性的潜在突变。作为突变体表型的一部分，腺苷酸环化酶对福斯高林的激活具有部分抗性，对促肾上腺皮质激素（ACTH）完全不敏感，对氟化钠（NaF）完全有反应；质膜组分中Gsα和G1α的水平降低；Gβ/γ的活性受损。在本研究中，我们研究了与福斯高林抗性相关的复杂表型的基础，以更好地理解有助于调节腺苷酸环化酶活性的因素。我们证明这些突变体对ACTH的抗性是由于未能表达ACTH受体转录本。用编码小鼠β2 - 肾上腺素能受体的基因转染这些突变体，导致转化体恢复正常的受体 - G蛋白偶联，并且Gsα和G1α的水平增加，接近亲本Y1细胞中的水平。然而，这些β2 - 肾上腺素能受体转化体对福斯高林和ACTH仍然具有抗性。两个自发的Y1突变体Y6和OS3，先前被表征为未能积累ACTH受体转录本的ACTH抗性克隆，被证明对福斯高林具有抗性，并且膜组分中含有较少的Gsα，这表明福斯高林抗性、未能表达ACTH受体以及随之而来的Gsα减少密切相关。人ACTH受体在Y6和OS3细胞中的表达恢复了ACTH反应性腺苷酸环化酶活性并增加了Gsα的水平，但并未逆转福斯高林抗性表型。总之，这些结果表明，对福斯高林抗性的突变具有下游后果，导致ACTH受体表达丧失以及随之而来的膜相关Gα亚基水平降低。结果进一步表明，G蛋白偶联受体可能对与细胞膜相关的Gα亚基具有稳定作用。根据当前模型，福斯高林通过与腺苷酸环化酶和Gsα形成三元复合物来激活腺苷酸环化酶。我们的结果表明该模型可能不完整，并且福斯高林对腺苷酸环化酶的最佳激活需要直接或间接起作用的额外成分。
