Changes in biological activity and folding of guanylate cyclase-activating protein 1 as a function of calcium.

Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity. The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly2-Lys9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1(E75D,E111D,E155D) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100beta, a high [Ca2+]free activator of GC1, competed with the triple mutant at high [Ca2+]free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.

Functional differences in the interaction of arrestin and its splice variant, p44, with rhodopsin.

Arrestin quenches signal transduction in rod photoreceptors by blocking the catalytic activity of photoactivated phosphorylated rhodopsin toward the G protein, transducin (Gt). Rod cells also express a splice variant of arrestin, termed p44, in which the last 35 amino acids are replaced by a single Ala. In contrast to arrestin, this protein has been reported to bind to both the phosphorylated and nonphosphorylated forms of the activated receptor. In this study, we analyzed formation of the rhodopsin-p44 complex in vitro. Like arrestin, p44 stabilized the meta II (MII) photoproduct relative to forms MI and MIII and did not interact measurably with the apoprotein opsin. However, several differences between p44 and its parent protein were found: (i) p44 binds to nonphosphorylated MII with a much lower affinity (KD = 0.24 microM) than to phosphorylated MII (P-MII) (KD = 12 nM); arrestin binds only to P-MII (KD = 20 nM); (ii) p44 interacted also with truncated MII (329G-Rho MII), which lacked the sites of phosphorylation; (iii) with both MII and P-MII, the activation energy of complex formation with p44 was lower than that found for arrestin (70 kJ/mol instead of 140 kJ/mol); and (iv) InsP6 inhibited poorly the interaction between p44 and P-MII, but it strongly inhibited the interaction between arrestin and P-MII. Extrapolation of the measured on-rates to physiological conditions yielded reaction times for the binding of p44 to activated rhodopsin. The data suggest that the splice variant, p44, and its parent protein, arrestin, play different roles in phototransduction. The physiological significance of these differences remains to be determined.

Functional reconstitution of photoreceptor guanylate cyclase with native and mutant forms of guanylate cyclase-activating protein 1.

In rod and cone photoreceptor cells, activation of particulate guanylate cyclase (retGC1) is mediated by a Ca2+-binding protein termed GCAP1, that detects changes in [Ca2+]free. In this study, we show that N-acylated GCAP1 restored Ca2+ sensitivity of native and recombinant photoreceptor retGC1. ATP increased the affinity of retGC1 for GCAP1 and accelerated catalysis. Using peptides derived from the GCAP1 sequence, we found that at least three regions, encompassing the N-terminus, the EF-1 motif, and the EF-3 motif, were likely involved in the interaction with retGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation at the N-terminus (delta25-GCAP1) reduced retGC1-stimulating activity dramatically, while deletion of 10 amino acids (delta10-GCAP1) reduced the specific activity by only approximately 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+]free, in conditions that inactivated native GCAP1, retGC1 showed significant activity in the presence of delta10-GCAP1. Native and all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluorescence. All mutants bound to ROS membranes in a Ca2+-independent manner, except delta25-GCAP1, which was mostly soluble. These findings suggest that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.

Calcium modulation of bovine photoreceptor guanylate cyclase.

Bovine photoreceptor guanylate cyclase (ROS-GC) consists of a single transmembrane polypeptide chain with extracellular and intracellular domains. In contrast to non-photoreceptor guanylate cyclases (GCs) which are activated by hormone peptides, ROS-GC is modulated in low Ca2+ by calmodulin-like Ca(2+)-binding proteins termed GCAPs (guanylate cyclase-activating proteins). In this communication we show that, like the native system, ROS-GC expressed in COS cells is activated 4-6-fold by recombinant GCAP1 at 10 nM Ca2+ and that the reconstituted system is inhibited at physiological levels of Ca2+ (1 microM). A mutant ROS-GC in which the extracellular domain was deleted was stimulated by GCAP1 indistinguishable from native ROS-GC indicating that this domain is not involved in Ca2+ modulation. Deletion of the intracellular kinase-like domain diminished the stimulation by GCAP1, indicating that this domain is at least in part involved in Ca2+ modulation. Replacement of the catalytic domain in a non-photoreceptor GC by the catalytic domain of ROS-GC yielded a chimeric GC that was sensitive to ANF/ATP and to a lesser extent to GCAP1. The results establish that GCAP1 acts at an intracellular domain, suggesting a mechanism of photoreceptor GC stimulation fundamentally distinct from hormone peptide stimulation of other cyclase receptors.

Structural and enzymatic aspects of rhodopsin phosphorylation.

Photoactivated rhodopsin (Rho*) is phosphorylated near the C terminus at multiple sites, predominantly at Ser334, Ser338, and Ser343. We systematically examined the sites of phosphorylation upon flash activation of Rho in rod outer segment (ROS) homogenates. Addition of an inhibitory antibody against rhodopsin kinase (RK) lowered phosphorylation at Ser334, Ser338, and Ser343, without changing the ratio between phosphorylation sites. In contrast, no effect of protein kinase C was detected after stimulation (by a phorbol ester), inhibition (with H7), or reconstitution of protein kinase C with purified ROS membranes. The stoichiometry and the ratio between different phosphorylation sites in purified Rho were also reproduced using RK, purified to apparent homogeneity from ROS or from an insect cell expression system. Thus, we conclude that light-dependent phosphorylation of Rho is mediated primarily by RK. Depalmitoylation of Rho at Cys322 and Cys323 altered the conformation of the C terminus of Rho, as observed by phosphorylation by casein kinase I, but did not affect phosphorylation by RK. The sites of phosphorylation were influenced, however, by the presence of four conserved amino acids at the C terminus of Rho. The accumulation of phosphorylated Ser334 observed in vivo could result from slower dephosphorylation of this site as compared with dephosphorylation of Ser338 and Ser343. These data provide a molecular mechanism for the site-specific phosphorylation of Rho observed in vivo.

Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer's disease.

We present the first evidence for carbonyl-related posttranslational modifications of neurofilaments in the neurofibrillary pathology of Alzheimer's disease (AD). Two distinct monoclonal antibodies that consistently labeled neurofibrillary tangles (NFTs), neuropil threads, and granulovacuolar degeneration in sections of AD tissue also labeled the neurofilaments within axons of the white matter following modification by reducing sugars, glutaraldehyde, formaldehyde, or malondialdehyde. The epitope recognized by these two antibodies shows a strict dependency for carbonyl modification of the neurofilament heavy subunit. The in vivo occurrence of this neurofilament modification in the neurofibrillary pathology of AD suggests that carbonyl modification is associated with a generalized cytoskeletal abnormality that may be critical in the pathogenesis of neurofibrillary pathology. Furthermore, the data presented here support the idea that extensive posttranslational modifications, including oxidative stress-type mechanisms, through the formation of cross-links, might account for the biochemical properties of NFTs and their resistance to degradation in vivo.

Inositolhexakisphosphate (InsP6): an antagonist of fibroblast growth factor receptor binding and activity.

Fibroblast growth factors (FGF), which have been implicated in tumor cell growth and angiogenesis, have biological activities that appear to be mediated by both heparinlike extracellular matrix sites and transmembrane tyrosine kinase receptor sites. In the present study, we demonstrated that inositolhexakisphosphate (InsP6) inhibits basic FGF (bFGF) binding to heparin. Our spectrofluorometric analyses demonstrated that InsP6 not only bound to bFGF, presumably within the bFGF heparin-binding domain, but also protected bFGF from degradation by trypsin. Also, InsP6 inhibited the cellular binding of bFGF and other fibroblast growth factor family members such as acidic FGF (aFGF) and K-FGF in a saturable and dose-dependent manner. Furthermore, concentrations as low as 100 microM InsP6 inhibited bFGF-induced DNA synthesis in AKR-2B fibroblasts, as well as the growth of bFGF- and K-FGF-transfected NIH/3T3 cells. Together, these results indicate that InsP6 may serve as a useful antagonist of FGF activity.