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Fig. 7. A model for the stimulatory effects by BPGAP1 and EEN on EGF-stimulated EGF receptor endocytosis and ERK1/2 phosphorylation. Stimulation of cells with EGF triggers the internalization of the EGF-bound EGF receptor in the form of vesicles, leading to the phosphorylation of ERK1/2. This process is enhanced by BPGAP1 or EEN alone and augmented through their interaction via their PRR and SH3 domain, respectively. However, the presence of NT mutant of EEN, which lacks the SH3 domain, completely prevents the effects of BPGAP1 in both endocytosis and ERK1/2 phosphorylation, thus acting as a dominant negative mutant in this pathway. This could involve displacing and sequestering functionally active EEN or other endophilins from BPGAP1, by forming homodimer or heterodimer complexes via their coiled-coil regions. Similarly, the GAP mutant R232A devoid of the catalytic arginine finger motif also reduces basal and EEN-stimulated EGF receptor endocytosis, suggesting that functional RhoGAP activity is required to promote endocytosis and ERK1/2 phosphorylation. Furthermore, despite failing to augment the EEN effects further and its inability to enhance endocytosis, the PP mutant (devoid of the interaction with EEN) or the GAP mutant (R232A) can also stimulate ERK1/2 phosphorylation, which suggests a distinct regulatory pathway independent of BPGAP1's stimulatory role in endocytosis. This could involve a different protein X possibly via its interaction with the BCH domain, but not linked to its direct interaction with EEN. However, this pathway still functions downstream of the control that requires an intact EEN function, as the NT mutant completely abolishes all stimulatory effect by BPGAP1, perhaps through a feedback or crosstalk from the endocytosis-dependent mechanism. BAR, BIN/amphiphysin/Rvsp domain; BCH, BNIP-2 and Cdc42GAP homology domain; GAP, GTPase-activating protein domain; PRR, proline-rich region; R232A, GAP mutant devoid of the catalytic arginine finger motif; NT, EEN mutant without the SH3 domain; ERK, extracellular signal-regulated kinase. Plus sign in circle denotes stimulatory effect while T-bars denote inhibitory effect. P, phosphorylation.
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