CRHR1 interaction with SAP97, SAP102 and PSD93 depends on the PDZ binding motif.

<p>Co-IPs were performed using lysates of HEK293 cells transiently transfected as indicated. (A) myc-GFP-CRHR1 but not myc-GFP-CRHR1STAVA was co-immunoprecipitated with HA-SAP97, and similarly, HA-SAP97 was co-immunoprecipitated with myc-GFP-CRHR1 but not with myc-GFP-CRHR1-STAVA. (B) HA-SAP97 PDZ1-3 and HA-SAP97 PDZ1-2 were co-immunoprecipitated with myc-GFP-CRHR1 and accordingly, these SAP97 variants were co-immunoprecipitated with myc-GFP-CRHR1 using an anti-myc antibody in the Co-IP. No interaction was observed for HA-SAP97 PDZ1. HA-SAP97 PDZ1-2 detected by the anti-HA antibody following the anti-myc Co-IP has the same molecular weight and thus is indistinguishable from the heavy chain of the anti-myc antibody (→). (C) myc-GFP-CRHR1 but not myc-GFP-CRHR1-STAVA was co-immunoprecipitated with flag-SAP102. Similarly, flag-SAP102 was co-immunoprecipitated with myc-GFP-CRHR1 but not with myc-GFP-CRHR1-STAVA. Moreover, flag-SAP102 PDZ3 was not detected following an immunoprecipitation (IP) of myc-GFP-CRHR1. (D) flag-CRHR1 but not flag-CRHR1-STAVA was co-immunoprecipitated with GFP-PSD93 and accordingly, GFP-PSD93 was co-immunoprecipitated with flag-CRHR1 but not with flag-CRHR1-STAVA. CRHR1 always showed high molecular weight complexes (*) together with the monomeric form (>). Therefore, high molecular weight complexes are also shown when necessary. Dashed lines indicate that the samples were run on the same immunoblot (IB), however, not in adjacent lanes. Continuous lines separate different IBs from the same experiment. (B) The ~55 kDa band represents the heavy chain of the primary antibody.