Heregulin-mediated activation of particular erbB receptor combinations was used as a model system to investigate the interaction of erbB3 and erbB4 with the adaptor protein growth factor receptor-bound (Grb)7. In human breast cancer cell lines, co-immunoprecipitation of Grb7 with both receptors was detected upon heregulin stimulation. This association was direct and mediated by the Grb7 Src homology (SH)2 domain. Co-expression of erbB2 with erbB3 point mutants was used to map Grb7 binding sites. This demonstrated that tyrosine 1180 and 1243 represent the major and minor sites of Grb7 interaction, respectively. Although these recognition sequences possess an Asn residue at +2 relative to the phosphotyrosine and therefore represent potential Grb2 binding sites, phosphopeptide competition and "pull-down" experiments demonstrated that they interact preferentially with the Grb7 versus the Grb2 SH2 domain. Substitution analysis indicated that an Arg residue at +3 could act as a selectivity determinant, but the effect was context-dependent. Consequently, the Grb2 and Grb7 SH2 domains possess overlapping, but distinct, specificities. These studies therefore identify Grb7 as an in vivo target of erbB3 and erbB4 and provide an underlying mechanism for the ability of erbB3 to recruit Grb7 and not Grb2, a property unique among erbB receptors.