Calcium binding, but not a calcium-myristoyl switch, controls the ability of guanylyl cyclase-activating protein GCAP-2 to regulate photoreceptor guanylyl cyclase.
Guanylyl cyclase-activating protein 2 (GCAP-2) is a recoverin-like calcium-binding protein that regulates photoreceptor guanylyl cyclase (RetGC) (Dizhoor, A. M., and Hurley, J. B. (1996) J. Biol. Chem. 271, 19346-19350). It was reported that myristoylation of a related protein, GCAP-1, was critical for its affinity for RetGC (Frins, S., Bonigk, W., Muller, F., Kellner, R., and Koch, K.-W. (1996) J. Biol. Chem. 271, 8022-8027). We demonstrate that the N terminus of GCAP-2, like those of other members of the recoverin family of Ca2+-binding proteins, is fatty acylated. However, unlike other proteins of this family, more GCAP-2 is present in the membrane fraction at low Ca2+ than at high Ca2+ concentrations. We investigated the role of the N-terminal fatty acyl residue in the ability of GCAP-2 to regulate RetGCs. Myristoylated or nonacylated GCAP-2 forms were expressed in Escherichia coli. Wild-type GCAP-2 and the Gly2 --> Ala2 GCAP-2 mutant, which is unable to undergo N-terminal myristoylation, were also expressed in mammalian HEK293 cells. We found that compartmentalization of GCAP-2 in photoreceptor outer segment membranes is Ca2+- and ionic strength-sensitive, but it does not require the presence of the fatty acyl group and does not necessarily directly reflect GCAP-2 interaction with RetGC. The lack of myristoylation does not significantly affect the ability of GCAP-2 to stimulate RetGC. Nor does it affect the ability of the Ca2+-loaded form of GCAP-2 to compete with the GCAP-2 mutant that constitutively activates RetGC. We conclude that while Ca2+ binding plays a major regulatory role in GCAP-2 function, it does not operate through a calcium-myristoyl switch similar to the one found in recoverin.
Olshevskaya
E V
EV
Department of Biochemistry and Howard Hughes Medical Institute, P.O. Box 357370, University of Washington, Seattle, Washington 98195-7370, USA.
Hughes
R E
RE
Hurley
J B
JB
Dizhoor
A M
AM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D004926:Escherichia coli; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D009226:Myristates; D010786:Photoreceptor Cells / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D009226:Myristates; D006162:Guanylate Cyclase; D002118:Calcium