Cross-talk between GPCR and RTK signaling pathways are crucial to the efficient relay and integration of cellular information. Here we identify and define the novel binding interaction of the E3 ubiquitin ligase AIP4 with the GTP exchange factor PIX. We demonstrate that this interaction is mediated in part by the PIX–SH3 domain binding to a proline-rich stretch of AIP4. Analysis of the interaction by isothermal calorimtery is consistent with a heterotrimeric complex with one AIP4 derived peptide binding to two PIX–SH3 domains. We determined the crystal structure of the PIX–SH3/AIP4 complex to 2.0 Å resolution. In contrast to the calorimetry results, the crystal structure shows a monomeric complex in which AIP4 peptide binds the PIX–SH3 domain as a canonical Class I ligand with an additional type II polyproline helix that makes extensive contacts with another face of PIX. Taken together, the novel interaction between AIP4 and PIX represents a new regulatory node for GPCR and RTK signal integration. Our structure of the PIX–SH3/AIP4 complex provides important insight into the mechanistic basis for PIX scaffolding of signaling components, especially those involved in cross-talk.