Localization of guanylate cyclase-activating protein 2 in mammalian retinas.

Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca2+, a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca2+-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.

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.

Mechanisms of opsin activation.

Rhodopsin is constrained in an inactive conformation by interactions with 11-cis-retinal including formation of a protonated Schiff base with Lys296. Upon photoisomerization, major structural rearrangements that involve protonation of the active site Glu113 and cytoplasmic acidic residues, including Glu134, lead to the formation of the active form of the receptor, metarhodopsin II b, which decays to opsin. However, an activated receptor may be generated without illumination by addition of all-trans-retinal or its analogues to opsin, as measured in this study by the increased phosphorylation of opsin by rhodopsin kinase. The potency of stimulation depended on the chemical and isomeric nature of the analogues and the length of the polyene chain with all-trans-C17 aldehyde and all-trans-retinal being the most active and trans-C12 aldehyde being the least active. Certain cis-isomers, 11-cis-13-demethyl-retinal and 9-cis-C17 aldehyde, were also active. Most of the retinal analogues tested did not regenerate a spectrally identifiable pigment, and many were incapable of Schiff base formation (ketone, stable oximes, and Schiff base-derivatives of retinal). Thus, receptor activation resulted from formation of non-covalent complexes with opsin. pH titrations suggested that an equilibrium exists between partially active (protonated) and inactive (deprotonated) forms of opsin. These findings are consistent with a model in which protonation of one or more cytoplasmic carboxyl groups of opsin is essential for activity. Upon addition of retinoids, the partially active conformation of opsin is converted to a more active intermediate similar to metarhodopsin II b. The model provides an understanding of the structural requirements for opsin activation and an interpretation of the observed activities of natural and experimental opsin mutants.

Expression of GCAP1 and GCAP2 in the retinal degeneration (rd) mutant chicken retina.

We cloned the guanylate cyclase activating proteins, GCAP1 and GCAP2, from chicken retina and examined their expression in normal and predegenerate rdlrd chicken retina. Northern analyses show that the amounts of the single transcripts encoding GCAP1 and GCAP2 are reduced to about 70% of normal levels in rdlrd retina. Western analyses reveal that GCAP2 levels appear normal in this retina, while GCAP1 levels are reduced by more than 90%. The specific downregulation of GCAP1 in rdlrd retina is consistent with a model for this disease in which activation of guanylate cyclase in the photoreceptors is abnormal, resulting in low levels of cGMP and an absence of phototransduction.

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.

Rod outer segment retinol dehydrogenase: substrate specificity and role in phototransduction.

The reaction catalyzed by all-trans-retinol dehydrogenase of rod outer segments completes the quenching of photoactivated rhodopsin and initiates the cycle of reactions leading to regeneration of visual pigment. The goal of this study was to determine the kinetic parameters of the dehydrogenase at physiological levels of bleaching, to investigate its specificity, and to determine its possible role in modulating phototransduction. Reduction of all-trans-retinal could be measured after bleaching < 0.15% rhodopsin. Kinetic parameters for the forward reaction determined with endogenous all-trans-retinal were Km = 1.1 microM; Vmax = 7 nmol/min/mg rhodopsin. The low enzymatic activity suggests that at high bleach rates, all-trans-retinal could accumulate, increasing the steady state level of bleaching intermediates or promoting formation of pseudophotoproducts. Active pseudophotoproducts, which stimulate Gt activation and opsin phosphorylation by rhodopsin kinase, are formed with opsin and all-trans-retinal as well as retinal analogues lacking the 13 methyl or the terminal two carbons of the polyene chain. Addition of all-trans-retinol, NADP, and [32P]ATP to rod outer segments increased rhodopsin phosphorylation. Kinetic parameters for the reverse reaction determined with exogenous all-trans-retinol were Km = 10 microM; Vmax = 11 nmol/min/mg rhodopsin. Our results support the hypothesis that all-trans-retinol dehydrogenase could influence the phototransduction cascade, including activities of Gt, rhodopsin kinase, and binding of arrestin, by impeding the recycling of rhodopsin at high bleach levels.

Opsins with mutations at the site of chromophore attachment constitutively activate transducin but are not phosphorylated by rhodopsin kinase.

More than 70 mutations in the gene encoding the visual pigment rhodopsin have been identified in patients with autosomal dominant retinitis pigmentosa. Most of these mutations are thought to interfere with proper folding of the membrane protein. However, families with a severe phenotype of retinitis pigmentosa have been identified and shown to carry a mutation at the site of chromophore attachment, Lys-296. This mutation disrupts the inactive conformation of opsin and results in a constitutively active protein that can activate the rod-specific GTP-binding protein, transducin, in the absence of light and in the absence of the chromophore 11-cis-retinal. It has been suggested that this mutant opsin molecule may cause rod degeneration by depletion of the components used to inactivate rhodopsin, such as rhodopsin kinase. In this work we test this idea by determining whether two constitutively active opsin mutants are phosphorylated by rhodopsin kinase. We found that opsin mutants where Lys-296 is replaced either by Glu (K296E) or by Gly (K296G) are not substrates of rhodopsin kinase in the absence of chromophore. However, when K296G is regenerated with a Schiff base complex of 11-cis-retinal and n-propylamine and exposed to illumination, phosphorylation of opsin occurs. These experiments suggest that in the rod photoreceptors of patients with retinitis pigmentosa carrying a mutation at Lys-296, there is persistent activation of the GTP-binding protein-mediated cascade. This may result in a situation that mimics long-term exposure to continuous illumination and results in the degeneration of photoreceptors.

Characterization of a truncated form of arrestin isolated from bovine rod outer segments.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.

Assays of retinoid dehydrogenases by phase partition.

Two modifications of an extraction assay for retinoid dehydrogenases are described. The first method involves the transfer of tritium from carbon-15 of [15-3H]-retinol or [15-3H]retinaldehyde to NAD, whereas in the second method, tritium from [3H]NADPH is transferred to all-trans-retinaldehyde. Since both versions of the assay involve the interconversion of water-soluble and -insoluble tritium-labeled compounds, a simple phase partition is sufficient to separate labeled products from labeled reactants. The assays are shown to provide reliable estimations of the reaction progress for three retinoid dehydrogenases of the visual system when compared to HPLC analysis of retinoid products or gel filtration analysis of pyridine nucleotide. The assays will be useful in studying retinoid dehydrogenases from other tissues and in principle can be modified for other dehydrogenase reactions with water-insoluble substrates.

Identification of the N-terminal region in rhodopsin kinase involved in its interaction with rhodopsin.

Upon illumination rhodopsin kinase (RK) phosphorylates the visual pigment, rhodopsin, in a reaction that is thought to terminate in part the biochemical events that follow photon absorption. In this paper, RK was studied to assign functional regions to the primary structure of the enzyme. Peptides derived from the sequence of RK were used to prepare site-specific anti-peptide antibodies against: 1) the N-terminal region located between residues 17 and 34, which contains an autophosphorylation site; 2) the Lys/Arg-rich region corresponding to residues 216-237 near the catalytic domain; 3) the region located between residues 483 and 497, which encompasses the major autophosphorylation sites; and 4) the C-terminal region located between residues 539 and 556, close to the isoprenylation site of RK. Antibodies also were raised against purified RK. Application of the antibodies directed against the N-terminal domain blocks RK activity toward Rho*, but has no affect on the phosphorylation of a synthetic peptide substrate. Additionally, a significant portion of the inhibitory effect seen with an antibody directed against whole RK could be reversed by the peptide derived from the N-terminal region. We conclude that the N-terminal region of RK contains a sequence involved in the recognition of photolyzed Rho. Furthermore, the inhibition of RK activity eliminates the effect of ATP during the inactivation of cGMP phosphodiesterase, implying that RK is a necessary component of a cascade of reactions involved in the quenching of phototransduction. Light microscopic immunocytochemistry using these antibodies revealed that RK was localized to the rod and cone outer segments of human and bovine retinas.

Identification of the autophosphorylation sites in rhodopsin kinase.

Rhodopsin kinase (RK) is a second-messenger-independent protein kinase that is involved in deactivation of photolyzed rhodopsin (Rho*). We have developed a significantly improved method for isolation of RK based on the specific interactions of phosphorylated forms of the enzyme with heparin-Sepharose. Conversion of the dephosphorylated form of RK to the fully phosphorylated enzyme leads to specific elution of the kinase from the resin. Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites. Peptides containing the autophosphorylation sites were isolated by reverse-phase high performance liquid chromatography and analyzed by Edman degradation and tandem mass spectrometry. The derived amino acid sequence of the peptide containing the major autophosphorylation site yielded the following sequence: DVGAFS488T489VKGVAFEK, where Ser488 and Thr489 are phosphorylated. Additionally, a minor autophosphorylation site was identified at Ser21. A 15-residue peptide (DVGAFSTVKGVAFEK) encompassing the major autophosphorylation site was synthesized and used for phosphorylation and inhibition studies. In contrast to many other protein kinases, the low catalytic activity of RK toward its autophosphorylation site peptide and the poor inhibitory properties of this peptide suggest unique properties of this member of the family of G protein-coupled receptor kinases.

The role of arrestin and retinoids in the regeneration pathway of rhodopsin.

Phototransduction results from a cascade of reactions that culminate in a neuronal signal. Photoisomerization of rhodopsin's chromophore, 11-cis-retinal to all-trans-retinal, leads to the formation of the activated photoproduct metarhodopsin II (Meta II). Subsequently, Meta II initiates the excitation events by activating many copies of the rod cell-specific G-proteins (Gt or transducin). To terminate the signal, the long-lived Meta II must be quenched. Deactivation of Meta II involves phosphorylation by rhodopsin kinase followed by the binding of arrestin. In order to recycle rhodopsin for phototransduction, arrestin must dissociate, and the chromophore must be replaced. In this study, we show that the reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol is essential for recycling photoactivated rhodopsin. Once this reduction has occurred, the arrestin blockade of the receptor is removed, the chromophore site becomes accessible for regeneration, and the phosphates can be hydrolyzed. If the reduction does not occur, we demonstrate that free all-trans-retinal can react with the apoprotein to form pseudo-photoproducts that are spectrally identical to the photoinduced metarhodopsin species (Meta I/II/III). The Meta II-like product, M380, interacts tightly with arrestin and kinase, however, it does not measurably interact with Gt. The persistent blockade by arrestin and the low affinity for Gt together prevent activation of the visual cascade. Therefore, any insufficiency in the reduction of all-trans-retinal to all-trans-retinol may lead to the accumulation of M380-arrestin in situ, which may effect adaptational processes.

Binding of inositol phosphates to arrestin.

Arrestin binds to phosphorylated rhodopsin in its light-activated form (metarhodopsin II), blocking thereby its interaction with the G-protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin-rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5-tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 microM and 5 microM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.

Phosphorylated rhodopsin and heparin induce similar conformational changes in arrestin.

Photoactivated rhodopsin is quenched upon its phosphorylation in the reaction catalyzed by rhodopsin kinase and the subsequent binding of a regulatory protein, arrestin. We have found that heparin and other polyanions compete with photoactivated, phosphorylated rhodopsin to bind arrestin (48-kDa protein, S-antigen). This is shown (a) by the suppression of stabilized metarhodopsin II; (b) by changes in the digestion of arrestin in the presence of heparin; and (c) by the restoration of arrestin-quenched phosphodiesterase activity. When bound to arrestin, heparin also mimics phosphorylated rhodopsin by similarly exposing arrestin to limited proteolysis. We conclude that heparin and rhodopsin have similar means of binding to arrestin, and we propose a cationic region of arrestin (beginning with Lys163 of the bovine sequence) as the interaction site. In agreement with previous kinetic data we interpret the results in terms of a binding conformation of arrestin which is stabilized by rhodopsin or heparin and is open to proteolytic attack.

Role of the carboxyl-terminal region of arrestin in binding to phosphorylated rhodopsin.

The structural and functional properties of arrestin were studied by subjecting the protein to limited proteolysis. Limited proteolysis by trypsin cleaves arrestin (48 kDa), producing 20-25-kDa fragments. Prior to this stage of proteolysis, trypsin produced 46.6-, 45.4-, and 42-kDa fragments. Structural analysis of the proteolytic fragments demonstrated major cleavage at the carboxyl terminus, indicating that the carboxyl terminus is highly exposed. We found that forms of arrestin truncated at their carboxyl terminus maintained their functional properties and bound to phosphorylated rhodopsin. Native arrestin binds only to photoexcited phosphorylated rhodopsin, whereas the truncated arrestin binds to phosphorylated rhodopsin independent of its exposure to light. The truncated forms of arrestin were separated from native arrestin by a chromatographic procedure and subsequently characterized in functional studies. The binding of the truncated forms of arrestin to phosphorylated photoexcited rhodopsin is more tight than the binding of native arrestin as determined by a direct binding assay and the phosphodiesterase assay. We suggest that the acidic carboxyl-terminal region of arrestin may act as a regulator for light-dependent binding to phosphorylated rhodopsin.

Mechanism of rhodopsin kinase activation.

The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.

Regulation of rhodopsin kinase by autophosphorylation.

Rhodopsin kinase (RK) catalyzes the phosphorylation of rhodopsin (Rho) as one of the steps in quenching photoactivated Rho. In this work, we investigated the autophosphorylation of RK and how it affects the interaction between RK and Rho. RK undergoes intramolecular phosphorylation, resulting in the incorporation of three or four phosphates per RK molecule. Phosphorylated RK subsequently is a substrate for protein phosphatases 2A and 2B. We isolated three forms of RK based on their differential interactions with heparin-Sepharose. Fully phosphorylated RK (alpha-RK) binds tightly to Rho but has significantly lower affinity to phosphorylated Rho, whereas unphosphorylated RK (gamma-RK) binds avidly to both forms of Rho. The heterogenous intermediately phosphorylated RK (beta-RK) was not studied. Our data support the hypothesis that RK dissociates from Rho when both Rho and RK become phosphorylated, thereby allowing the binding of arrestin to phosphorylated Rho. These results suggest that autophosphorylation plays an important role in regulating the binding of RK to Rho and that the binding sites of RK and arrestin overlap at least partially.

A photoreceptor calcium binding protein is recognized by autoantibodies obtained from patients with cancer-associated retinopathy.

Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity-purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure.

Reversible denaturation with urea of rabbit liver fructose-1,6-bisphosphatase.

Denaturation of fructose-1,6-bisphosphatase (Fru-P2-ase, EC 3.1.3.11) by urea and renaturation of denatured enzyme has been investigated. Denaturation lowers the specific activity of the enzyme but even at 8 M urea concentration in the presence of sucrose the activity of the enzyme is detectable. Centrifugation of the enzyme in a sucrose density gradient at 4 M urea reveals one peak of protein corresponding to a dimer. Denaturation increases intensity of intrinsic fluorescence of Fru-P2-ase and causes a red shift of fluorescence peak of the thioisoindole derivative of the enzyme. Renaturation of the denatured enzyme followed as the reappearance of enzymatic activity in the presence and absence of bovine serum albumin (BSA) is characterised by first order kinetics, k = 1.78 X 10(-3) s-1. The presence of BSA does not affect the rate of renaturation but perceptibly increases the recovery of enzymatic activity. A 100% recovery of Fru-P2-ase activity is observed at 0.5 micrograms/mL concentration of the enzyme and 2 mg/mL of BSA.

Structural changes of rabbit liver fructose-1,6-bisphosphatase: the effect of urea and subtilisin digestion.

At pH 6.3 both the native and subtilisin-digested fructose-1,6-bisphosphatase (Fru-P2-ase) molecules exhibit four fast-reacting thiol groups. The kinetic analysis shows that the pK value for the reaction of these thiols is 8.1. The increase of pH from 6.3 to 9.3 results in an uncovering of the remaining 20 thiol groups. In subtilisin-cleaved enzyme the rate of reaction of SH groups is considerably higher than in the native enzyme at pH 9.3, indicating changes in the microenvironments around thiols upon modification. A fluorescent label inserted on a fast-reacting SH group and neighboring NH2 group shifts the pH optimum of the enzyme to alkaline region and decreases its sensitivity toward AMP. Spectral analysis of labeled enzyme indicates that the labeled region of protein is more hydrophilic upon proteolytic digestion. It is concluded that a molecule of subtilisin-digested enzyme has a more relaxed structure than the native enzyme. The relaxation of the enzyme to a new conformation is reflected by urea addition, which mimics the effect of subtilisin digestion. Correlation of enzyme activity versus its sensitivity toward AMP (I 0.5), shows that at low concentrations of urea the active-site region at pH 6.3 is more affected than the region of AMP binding.