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 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.
Tucker
C L
CL
Howard Hughes Medical Institute and Department of Biochemistry, Box 357370, University of Washington, Seattle, Washington 98195, USA.
Laura
R P
RP
Hurley
J B
JB
D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D004795:Enzyme Stability; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000201:enzymology*
D000227:Adenine Nucleotides; D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D009419:Nerve Tissue Proteins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase