Independent regulation of Rap1 and mitogen-activated protein kinase by the alpha chain of Go.
Receptors coupled to G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) cascade. The intracellular pathways linking the alpha chains of these G proteins to MAPK activation are not completely understood. One of the signaling molecules which has been suggested to act downstream of Galpha(i/o) is the small G protein Rap1. We investigated the role of Rap1 in MAPK stimulation by Galpha(o) in Chinese hamster ovary (CHO) cells. Our previous results have shown that in this cell system activated Galpha(o) strongly potentiates the MAPK response to the epidermal growth factor (EGF) receptor. Rap1 regulation was examined in cells transfected with Rap1 and wild-type Galpha(o) or the activated mutant Galpha(o)-Q205L. Immunocytochemical analysis detected both Rap1 and the Galpha(o) subunit at the plasma membrane as well as on perinuclear cytoplasmic vesicles. Expression of wild-type Galpha(o) had no significant effect on the levels of activated Rap1. In contrast, Galpha(o)-Q205L virtually abolished the activation of Rap1 induced by EGF. Further experiments showed that MAPK stimulation by EGF was greatly inhibited by expression of activated Rap1, suggesting that Rap1 inhibition could mediate the effect of Galpha(o) on the MAPK cascade. However, Galpha(o)-Q205L efficiently inhibited the activation of Rap1 induced by fibroblast growth factor (FGF). We have previously found that the ability of FGF to activate MAPK is not modified by Galpha(o). In addition, expression of the GAP protein RAP1GAPII blocked Rap1 activation without affecting EGF- or FGF-dependent MAPK stimulation. These findings provide evidence for independent regulation of Rap1 and MAPK by the G(o )alpha chain.
Bernasconi
Francesca
F
Department of Pharmacology, University of Milan, Milan, Italy.
Malgaroli
Antonio
A
Vallar
Lucia
L
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D022162:Cytoplasmic Vesicles / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D009154:Mutation / Q000235:genetics; D014162:Transfection; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D004815:Epidermal Growth Factor; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020827:rap1 GTP-Binding Proteins