Asunto(s)
Corteza Cerebral/química , Proteínas de Unión al GTP Heterotriméricas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida/métodos , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Datos de Secuencia Molecular , Subunidades de Proteína , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosAsunto(s)
Proteínas de Unión al GTP Heterotriméricas/química , Transducina/química , Secuencia de Aminoácidos , Animales , Química Encefálica , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Indicadores y Reactivos , Datos de Secuencia Molecular , Subunidades de Proteína , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Transducina/aislamiento & purificaciónRESUMEN
We characterized the variable processing of the G protein gamma subunit isoforms associated with bovine brain G proteins, a primary mediator of cellular communication. Ggamma subunits were isolated from purified brain G proteins and characterized by Edman sequencing, by MALDI MS, by chemical and/or enzymatic fragmentation assayed by MALDI MS, and by MS/MS fragmentation and sequencing. Multiple forms of six different Ggamma isoforms were detected. Significant variation in processing was found at both the amino termini and particularly the carboxyl termini of the proteins. All Ggamma isoforms contain a carboxyl-terminal CAAX motif for prenylation, carboxyl-terminal proteolysis, and carboxymethylation. Characterization of these proteins indicates significant variability in the normal processing of all of these steps in the prenylation reaction, including a new variation of prenyl processing resulting from cysteinylation of the carboxyl terminus. These results have multiple implications for intracellular signaling mechanisms by G proteins, for the role of prenyl processing variation in cell signaling, and for the site of action and consequences of drugs that target the prenylation modification.
Asunto(s)
Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Isoformas de Proteínas/metabolismo , Proteómica , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Subunidades gamma de la Proteína de Unión al GTP/química , Isoformas de Proteínas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Heterotrimeric G proteins transduce signals from activated transmembrane G protein-coupled receptors to appropriate downstream effectors within cells. Signaling specificity is achieved in part by the specific alpha, beta, and gamma subunits that compose a given heterotrimer. Additional structural and functional diversity in these subunits is generated at the level of posttranslational modification, offering alternate regulatory mechanisms for G protein signaling. Presented here is the identification of a variant of the gamma(2) subunit of G protein heterotrimer purified from bovine brain and the demonstration that this RDTASIA gamma(2) variant, containing unique amino acid sequence at its N terminus, is a substrate for ubiquitylation and degradation via the N-end rule pathway. Although N-end-dependent degradation has been shown to have important functions in peptide import, chromosome segregation, angiogenesis, and cardiovascular development, the identification of cellular substrates in mammalian systems has remained elusive. The isolation of RDTASIA gamma(2) from a native tissue represents identification of a mammalian N-end rule substrate from a physiological source, and elucidates a mechanism for the targeting of G protein gamma subunits for ubiquitylation and degradation.