The scaffold protein CNK1 mediates proliferative as well as antiproliferative responses including differentiation and apoptosis. The angiotensin II type 2 (AT(2)) receptor belongs to the class of G protein-coupled receptors and also promotes antiproliferative effects. Here we report that CNK1 binds through the sterile alpha motif (SAM) and the conserved region in CNK (CRIC) to the AT(2) receptor. The exchange of a conserved leucine residue with arginine in the CRIC domain increases the binding affinity of CNK1 to the AT(2) receptor. The insertion ofa negatively charged amino acid stretch into the linker region between the N- and the C-terminal part ofCNKI strengthens the interaction between CNK1 and the AT(2) receptor in a Ras-regulated manner. The biological significance of the interaction was supported by coprecipitation of CNK1 and the AT(2) receptor in mouse heart extracts. Thus, CNK1 may play a role in the AT(2) receptor-mediated signaling pathways. (c) 2005 Elsevier Inc. All rights reserved.