The D2 dopamine receptor isoforms signal through distinct Gi alpha proteins to inhibit adenylyl cyclase. A study with site-directed mutant Gi alpha proteins.
The D2 dopamine receptor isoforms signal through distinct Gi alpha proteins to inhibit adenylyl cyclase. A study with site-directed mutant Gi alpha proteins.
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发表时间：
1994-09
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通讯作者：
S. Senogles
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作者：
S. Senogles
构建Gi1 α、Gi21 α和Gi3 α的cDNA的定点突变，其将C末端的半胱氨酸残基改变为甘氨酸残基（Gi α PT）。Gi α的这种突变不允许随后通过百日咳毒素进行共价修饰，这需要半胱氨酸部分。将每个突变体Gi α亚基的cDNA转染到具有D2多巴胺受体的任一种选择性剪接形式的GH4C1细胞中，并产生克隆系。在用百日咳毒素处理以去除来自内源性Gi蛋白的贡献后，检查腺苷酸环化酶的受体介导的抑制。D2多巴胺受体，短型（D2s）在这些细胞中通过Gi2 α PT突变体发出信号，其对激动剂的亲和力与在用单独的D2s的cDNA转染的细胞中观察到的亲和力或在不存在百日咳毒素的情况下观察到的信号传导相当。长形式的D2多巴胺受体（D2l）通过Gi3 α PT突变体发出信号以抑制毛喉素刺激的腺苷酸环化酶，其对激动剂的亲和力与在用单独的D2l的cDNA转染的细胞中观察到的亲和力相当。生长抑素（生长激素释放抑制因子）的受体被用作这些细胞系中的内源性对照受体。生长激素释放抑制因子能够通过Gi1 α PT和Gi3 α PT发出信号，以抑制毛喉素刺激的腺苷酸环化酶。这些结果表明，受体使用不同的Gi蛋白向共同的效应物发出信号。
Site-directed mutations of the cDNA for Gi1 alpha, Gi21 alpha, and Gi3 alpha were constructed which changed the cysteine residue at the C terminus to a glycine residue (Gi alpha PT). This mutation of the Gi alpha would not permit the subsequent covalent modification by pertussis toxin, which requires the cysteine moiety. The cDNA for each of the mutant Gi alpha subunits was transfected into GH4C1 cells with either of the alternative splice forms of the D2 dopamine receptor and clonal lines were generated. After treatment with pertussis toxin to remove the contribution from endogenous Gi proteins, the receptor-mediated inhibition of adenylyl cyclase was examined. The D2 dopamine receptor, short form (D2s) signaled through the Gi2 alpha PT mutant in these cells with an affinity for agonist which was comparable to that observed in cells transfected with the cDNA for D2s alone or the signaling observed in the absence of pertussis toxin. The long form of the D2 dopamine receptor (D2l) signaled through the Gi3 alpha PT mutant to inhibit forskolin-stimulated adenylyl cyclase, with an affinity for agonist comparable to that observed in cells transfected with the cDNA for D2l alone. The receptor for somatostatin (somatotropin release inhibiting factor) was used as an endogenous control receptor in these cell lines. The somatotropin release inhibiting factor was able to signal through both Gi1 alpha PT and Gi3 alpha PT to inhibit forskolin-stimulated adenylyl cyclase. These results indicated that receptors use distinct Gi proteins to signal to a common effector.