Figure 5.

Mapping of the binding determinants involved in the interactions between GPR179, R9AP, and RGS proteins. HEK293 cells were cotransfected with the indicated constructs, and coimmunoprecipitation assays were conducted. A, Schematic representation of GPR179 and R9AP organization with the annotation of the protein fragments deleted in the various constructs used in the binding experiments. B, Deletional mutagenesis of R9AP binding determinants in GPR179. An antibody against the myc-tagged deletion mutants of GPR179 was used for immunoprecipitation. R9AP was detected only after coimmunoprecipitation with GPR179-ΔCT2. C, D, Deletional mutagenesis of RGS11 and RGS7 binding determinants in GPR179, respectively. An antibody against the myc-tagged deletion mutants of GPR179 was used for immunoprecipitation. RGS11 (C) and RGS7 (D) were coimmunoprecipitated with GPR179-ΔCT2 and GPR179-ΔCT1 constructs. E, Deletional mutagenesis of GPR179 binding determinants in R9AP. An antibody against R9AP was used in the experiment. Deletion of the transmembrane region of R9AP does not affect its interaction with GPR179. F, Coimmunoprecipitation of R9AP and GPR179 deletional mutants containing minimal binding determinants. Asterisk indicates the position of nonspecific band detected in the immunoprecipitation eluates, which likely corresponds to IgGs used for affinity precipitation.