| Nerve growth factor (NGF) elicits the differentiation of PC12 cells through the sustained activation of the MAP Kinases, ERK 1 and 2. NGF binds its receptor, Trk A, which rapidly associates with multiple effector proteins, leading to the activation of the ERKs through a signaling cascade employing the small G proteins Ras and Rap 1. Upon activation, Trk A associates with cholesterol-rich lipid rafts, resulting in its interaction with critical signaling molecules contained within these membrane microdomains. We have investigated the mechanism by which Trk A concentrates in lipid rafts following NGF stimulation. Disruption of cholesterol-rich domains using methyl-beta-cyclodextrin (MbetaCD) attenuates ERK 1 and 2 activation in response to NGF stimulation, demonstrating that lipid rafts are essential for their activation. The concentration of Trk A within lipid raft domains is dependent on the c-Cbl associated protein (CAP). Following NGF stimulation, CAP associates with the guanine nucleotide exchange factor SOS. CAP is constitutively bound to the adaptor protein, APS, which associates with Trk A after NGF treatment. Introduction of a CAP mutant, CAPDeltaSoHo, which lacks a specific protein interaction domain for the lipid raft constituent, flotillin, inhibits the NGFstimulated concentration of Trk A and SOS to lipid raft domains. CAPDeltaSoHo also perturbs the activation of Ras and Rap 1, resulting in the suppression of ERK activation. These data provide a mechanism for the lipid raft localization of Trk A that is dependent on the CAP adaptor protein and establish the importance of lipid raft microdomains to ERK 1 and 2 signaling and neuronal differentiation. |
