DOMAIN-SPECIFIC STABILIZATION OF PHOTORECEPTOR MEMBRANE GUANYLYL CYCLASE BY ADENINE-NUCLEOTIDES AND GUANYLYL CYCLASE-ACTIVATING PROTEINS (GCAPS)

In photoreceptor outer segments, particulate guanylyl cyclase (RetGC) is stimulated at low intracellular Ca2+ concentrations by guanylyl cyc lase activating protein (GCAP), a Ca2+-sensitive activator, to resynth esize light-depleted cGMP. In washed outer segment membranes, we find that GCAP-stimulable RetGC is rapidly inactivated at physiological tem peratures (30-37 degrees C). Under the same conditions, RetGC remains competent for stimulation by S-100 protein preparations or Mn2+/Triton X-100, indicating that the cyclase catalytic domain remains functiona l. GCAPs and adenine nucleotides protect against inactivation. Protect ion by GCAPs is independent of Ca2+ concentration, suggesting that the re is a Ca2+-independent interaction between GCAP and RetGC. Protectio n by ATP (EC50 = 150 mu M) is not due to phosphorylation, since the no nhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP) protects eq ually well. In addition to their roles in protection, ATP and AMP-PNP also slowly stimulate cyclase activity. In parallel with the functiona l change in RetGC at physiological temperatures, we also observe a str uctural change. A 62-kDa intracellular fragment of RetGC-1 becomes mor e sensitive to cleavage by trypsin after preincubation at 30 degrees C unless ATP, AMP-PNP, or GCAP is present. Adenine nucleotides and GCAP s thus protect RetGC structurally, as well as functionally.