Santos Edson L, Reis Rosana I, Silva Ronaldo G, Shimuta Suma I, Pecher Christiane, Bascands Jean-Loup, Schanstra Joost P, Oliveira Laerte, Bader Michael, Paiva Antonio C M, Costa-Neto Claudio M, Pesquero João B
Department of Biophysics, Escola Paulista de Medicina, Federal University of São Paulo, 04023-062 São Paulo, SP, Brazil.
Regul Pept. 2007 Jun 7;141(1-3):159-67. doi: 10.1016/j.regpep.2006.12.030. Epub 2007 Jan 20.
Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.
早期对G蛋白偶联受体家族成员Mas原癌基因的研究提出，该基因编码一种功能性血管紧张素II（AngII）受体，然而进一步的研究结果并未证实这一假设。在本研究中，我们探讨了异二聚体AT(1)/Mas可能导致两种受体之间发生功能相互作用的假说。为此，用野生型AT(1)受体、无功能的AT(1)受体双突变体（C18F-K20A）和Mas，或用野生型/Mas和C18F-K20A/Mas转染CHO或COS-7细胞。单独表达Mas或C18F/K20A的细胞对AngII没有任何结合。野生型AT(1)受体和Mas的共表达显示出与单独表达野生型AT(1)时相似的结合模式。令人惊讶的是，双突变体C18F/K20A和Mas的共表达使AngII的结合亲和力完全恢复到与野生型AT(1)相似的水平。使用肌醇磷酸和细胞外酸化率测定的功能测量也显示，在共表达突变体C18F/K20A和Mas的细胞上，AngII的活性明显恢复。此外，免疫荧光分析表明，AT(1)受体主要定位于质膜，而突变体C18F-K20A仅定位于细胞内。然而，C18F-K20A突变体与Mas的共表达改变了突变体的分布模式，在质膜上出现强烈信号，与表达野生型AT(1)受体的细胞中观察到的信号相当。这些结果支持了Mas能够通过二聚化挽救缺陷型C18F-K20A突变体的结合和功能的假说。
