Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4.
J Neurochem
; 85(5): 1289-98, 2003 Jun.
Article
em En
| MEDLINE
| ID: mdl-12753087
ABSTRACT
Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha 2A-adrenoceptor and a pertussis toxin-resistant mutant of Go1 alpha. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha 2A-adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha 2A-adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1 alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1 alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.
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Base de dados:
MEDLINE
Assunto principal:
Ácidos Palmíticos
/
Proteínas Recombinantes de Fusão
/
Receptores Adrenérgicos alfa 2
/
Proteínas Heterotriméricas de Ligação ao GTP
/
Proteínas Ativadoras de GTPase
/
Proteínas RGS
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2003
Tipo de documento:
Article