THE MEMBRANE GUANYLYL CYCLASE, RETINAL GUANYLYL CYCLASE-1, IS ACTIVATED THROUGH ITS INTRACELLULAR DOMAIN

Retinal guanylyl cyclase-1 (RetGC-1) is a membrane guanylyl cyclase fo und in photoreceptor outer segments. It consists of an apparent extrac ellular domain (ECD) linked by a single transmembrane segment to an in tracellular domain (ICD). Guanylyl cyclase activating protein-2 (GCAP- 2) is a Ca2+-binding protein that activates RetGC-1 in a Ca2+-sensitiv e manner. To establish whether GCAP-2 stimulates RetGC-1 through the E CD or ICD, we made deletion mutants lacking either the ECD or both the ECD and transmembrane domains (TMD) of RetGC-1, Recombinant wild type RetGC-1 and both deletion mutants were expressed in HEK 293 cells, an d their sensitivities to GCAP-2, Ca2+, and ATP were compared. Our data demonstrate that both deletion mutants are regulated similarly to wil d type RetGC-1 with indistinguishable EC(50) values for Ca2+ and simil ar K-1/2 values for activation by GCAP-2, This shows that GCAP-2 funct ions through the ICD of RetGC-1 and that removal of the ECD and TMD do not significantly alter regulation by these factors. Our data also sh ow that ATP potentiates stimulation of guanylyl cyclase activity by GC AP-2 and that neither the ECD nor the TMD of RetGC-1 participate in it s regulation by ATP.