Calcium Binding, but Not a Calcium-Myristoyl Switch, Controls the Ability of Guanylyl Cyclase-activating Protein GCAP-2 to Regulate Photoreceptor Guanylyl Cyclase

Abstract

We demonstrate that the N terminus of GCAP-2, like those of other members of the recoverin family of Ca 2؉-binding proteins, is fatty acylated. However, unlike other proteins of this family, more GCAP-2 is present in the membrane fraction at low Ca 2؉ than at high Ca 2؉ 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 Gly 2 3 Ala 2 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 Ca 2؉ -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 Ca 2؉ -loaded form of GCAP-2 to compete with the GCAP-2 mutant that constitutively activates RetGC. We conclude that while Ca 2؉ 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.

Differential Regulation of CaV2.1 Channels by Calcium-Binding Protein 1 and Visinin-Like Protein-2 Requires N-Terminal Myristoylation

“…Effect of myristoylation of CaBP1 on voltage-dependent activation of Ca V 2.1 channels To investigate the functional role of myristoylation of CaBP1, we used a mutant, CaBP1/G2A, in which the glycine that is normally myristoylated has been mutated to alanine, a mutation that is known to prevent N-terminal myristoylation of proteins (Rocque et al, 1993;Olshevskaya et al, 1997). We began by characterizing the voltage dependence of Ca V 2.1 activation in cells cotransfected with wild-type or myristoyl-deficient CaBP1.…”

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

“…Recombinant myristoylated bovine and mouse GCAP-2, GCAP-1, and neurocalcin were expressed from pET11d vector (Novagen/EMD) in a BLR(DE3)pLysS Escherichia coli strain harboring yeast N-myristoyltransferase and purified as described previously in full detail (12,16,17).…”

“…Photoreceptor outer segment membranes were isolated under infrared illumination from frozen dark-adapted bovine retinas (Lawson and Lawson) using sucrose density gradient centrifugation and were washed in low salt buffer to remove endogenous GCAPs as described previously (12,16). Soluble Retinal Extract (S 100 )-Fresh bovine eyes obtained at a local slaughterhouse were dark-adapted for 2 h on ice, dissected under infrared light, and homogenized in 0.5 ml of buffer A (5 mM sodium phosphate buffer, pH 7.2, 50 mM NaCl) per retina on ice by 10 strokes in a Dounce homogenizer.…”

“…Phospho-GCAP-2 appears to be the first PP2C-specific substrate identified in photoreceptors. The only covalent modification in GCAP-2 isolated from bovine retinas was fatty acylation (16), and the presence of potent phosphatase PP2C in retinal extract (30) may account for the lack of phosphorylated GCAP-2 after its isolation from the retina. Therefore, it remains unclear to what extent GCAP-2 can be phosphorylated in vivo.…”

“…1, D and F). To minimize potential interference from phosphorylation of the endogenous GCAP-2, we extracted soluble retinal proteins (S 100 fraction) at normal ionic strength when GCAP-2 remained mostly associated with the membrane fraction (16). Although the S 100 fraction contains an ϳ33-kDa endogenous protein (presumably phosducin) that is highly phosphorylated by the CNDPK (27,29), this band migrates substantially higher than GCAP-2 and therefore does not interfere with the analysis.…”