Domain-specific stabilization of photoreceptor membrane guanylyl cyclase by adenine nucleotides and guanylyl cyclase activating proteins (GCAPs).

Abstract

In photoreceptor outer segments, particulate guanylyl cyclase (RetGC) is stimulated at low intracellular Ca2+ concentrations by guanylyl cyclase activating protein (GCAP), a Ca2+-sensitive activator, to resynthesize light-depleted cGMP. In washed outer segment membranes, we find that GCAP-stimulable RetGC is rapidly inactivated at physiological temperatures (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 functional. GCAPs and adenine nucleotides protect against inactivation. Protection by GCAPs is independent of Ca2+ concentration, suggesting that there is a Ca2+-independent interaction between GCAP and RetGC. Protection by ATP (EC50 = 150 microM) is not due to phosphorylation, since the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP) protects equally well. In addition to their roles in protection, ATP and AMP-PNP also slowly stimulate cyclase activity. In parallel with the functional change in RetGC at physiological temperatures, we also observe a structural change. A 62-kDa intracellular fragment of RetGC-1 becomes more sensitive to cleavage by trypsin after preincubation at 30 degrees C unless ATP, AMP-PNP, or GCAP is present. Adenine nucleotides and GCAPs thus protect RetGC structurally, as well as functionally.