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 found in photoreceptor outer segments. It consists of an apparent extracellular domain (ECD) linked by a single transmembrane segment to an intracellular domain (ICD). Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-binding protein that activates RetGC-1 in a Ca2+-sensitive manner. To establish whether GCAP-2 stimulates RetGC-1 through the ECD 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, and their sensitivities to GCAP-2, Ca2+, and ATP were compared. Our data demonstrate that both deletion mutants are regulated similarly to wild type RetGC-1 with indistinguishable EC50 values for Ca2+ and similar K1/2 values for activation by GCAP-2. This shows that GCAP-2 functions 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 show that ATP potentiates stimulation of guanylyl cyclase activity by GCAP-2 and that neither the ECD nor the TMD of RetGC-1 participate in its regulation by ATP.
Laura
R P
RP
Department of Biochemistry, University of Washington, Seattle, 98195-7370, USA.
Dizhoor
A M
AM
Hurley
J B
JB
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D008969:Molecular Sequence Data; D011817:Rabbits; D012160:Retina / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D002118:Calcium