Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene.
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.
Santos
Edson L
EL
Department of Biophysics, Escola Paulista de Medicina, Federal University of São Paulo, 04023-062 São Paulo, SP, Brazil.
Reis
Rosana I
RI
Silva
Ronaldo G
RG
Shimuta
Suma I
SI
Pecher
Christiane
C
Bascands
Jean-Loup
JL
Schanstra
Joost P
JP
Oliveira
Laerte
L
Bader
Michael
M
Paiva
Antonio C M
AC
Costa-Neto
Claudio M
CM
Pesquero
João B
JB
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D005452:Fluoresceins; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D007211:Indoles; D020128:Inhibitory Concentration 50; D007295:Inositol Phosphates / Q000032:analysis / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011519:Proto-Oncogenes / Q000235:genetics*; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007211:Indoles; D007295:Inositol Phosphates; D044140:Receptor, Angiotensin, Type 1; D043562:Receptors, G-Protein-Coupled; D000804:Angiotensin II; C007293:DAPI