Phospholipase C beta 1b (PLC beta 1b) is an atypical splice variant of PLC beta 1 that has a C-terminal proline-rich sequence instead of the PDZ-interacting motif common to other PLC beta subtypes. PLC beta 1b targets to the cardiomyocyte sarcolemma through an undefined association with the scaffolding protein Shank3. The C-terminal splice variant specific sequence of PLC beta 1b bound to deletion mutants of Shank3 that included the SH3 domain, but not to constructs lacking this domain. Mutating proline residues in the extreme C-terminal region of PLC beta 1b prevented the interaction between PLC beta 1b and Shank3 resulting in reduced sarcolemmal localization and downstream signalling responses. We conclude that PLC beta 1b activation and downstream signalling require the association of a previously unidentified C-terminal proline-rich motif with the SH3 domain of Shank3. PLC beta 1b is the first confirmed protein ligand for the SH3 domain of Shank3. (C) 2015 Elsevier Inc. All rights reserved.