Targeting of the small GTPase Rap2b, but not Rap1b, to lipid rafts is promoted by palmitoylation at Cys176 and Cys177 and is required for efficient protein activation in human platelets.
Rap1b and Rap2b are the only members of the Rap family of GTPases expressed in circulating human platelets. Rap1b is involved in the inside-out activation of integrins, while the role of Rap2b is still poorly understood. In this work, we investigated the localization of Rap proteins to specific microdomains of plasma membrane called lipid rafts, implicated in signal transduction. We found that Rap1b was not associated to lipid rafts in resting platelets, and did not translocate to these microdomains in stimulated cells. By contrast, about 20% of Rap2b constitutively associated to lipid rafts, and this percentage did not increase upon platelet stimulation. Rap2b interaction with lipid rafts also occurred in transfected HEK293T cell. Upon metabolic labelling with [(3)H]palmitate, incorporation of the label into Rap2b was observed. Palmitoylation of Rap2b did not occur when Cys176 or Cys177 were mutated to serine, or when the C-terminal CAAX motif was deleted. Contrary to CAAX deletion, Cys176 and Cys177 substitution did not alter the membrane localization of Rap2b, however, relocation of the mutants within lipid rafts was completely prevented. In intact platelets, disruption of Rap2b interaction with lipid rafts obtained by cholesterol depletion caused a significant inhibition of aggregation. Importantly, agonist-induced activation of Rap2b was concomitantly severely impaired. These results demonstrate that Rap2b, but not the more abundant Rap1b, is associated to lipid rafts in human platelets. This interaction is supported by palmitoylation of Rap2b, and is important for a complete agonist-induced activation of this GTPase.
Canobbio
Ilaria
I
Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy.
Trionfini
Piera
P
Guidetti
Gianni F
GF
Balduini
Cesare
C
Torti
Mauro
M
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002460:Cell Line; D002784:Cholesterol / Q000172:deficiency; D003545:Cysteine / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D054878:Lipoylation / Q000187:drug effects; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D013347:Subcellular Fractions / Q000378:metabolism; D020668:rap GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
D003902:Detergents; D002784:Cholesterol; C116753:RAP1B protein, human; C065562:RAP2B protein, human; D020559:Monomeric GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D003545:Cysteine