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PubMed1992

Two-site high-affinity interaction between inhibitory and catalytic subunits of rod cyclic GMP phosphodiesterase.

Light-activated cyclic GMP-phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells which regulates the level of the internal transmitter cyclic GMP. PDE consists of catalytic P alpha and P beta subunits, and two copies of inhibitory P gamma subunit. The two P gamma subunits block the enzyme's activity in the dark and are removed by the alpha-subunit of transducin (alpha 1) upon light-activation of photoreceptor cells. Here we have examined the role of various regions of P gamma, the N-terminal, the central cationic and the C-terminal regions, in interaction with the catalytic subunits of PDE. N-Terminal truncation of P gamma (12-87-P gamma) did not change the potency of PDE inhibition, and thus we conclude that the P gamma N-terminal region is not critical for P gamma-P alpha beta interaction. The central region, 24-46-P gamma, participates in interaction with the catalytic P alpha beta subunits. A synthetic peptide corresponding to this site inhibited approximately 50% of trypsin-activated PDE (tPDE) (Ki approximately 15 microM) and competed with P gamma for inhibition of tPDE. We demonstrated, by using h.p.l.c. gel filtration, that 125I-Tyr-24-46-P gamma peptide bound with high affinity to tPDE, but not to P alpha beta gamma 2. The C-terminal region of 46-87-P gamma was found to be the major region involved in inhibition of PDE. It fully inhibited tPDE with a Ki of approximately 0.8 microM. It also bound to tPDE, but not P alpha beta gamma 2, in h.p.l.c. gel-filtration experiments. In addition, P gamma was cross-linked by p-phenylenedimaleimide to both P alpha and P beta, as was shown by using subunit-specific anti-P alpha, -P beta and -P gamma antibodies. Cys68 of P gamma, which presumably participates in cross-linking, is located near the P gamma C-terminus. These data provide evidence for two regions of P gamma that interact with, and inhibit, P alpha beta. The central region, 24-46 P gamma, is important in binding, but inhibits PDE only weakly, whereas the C-terminal region is most important for PDE inhibition. These results help to explain the well-known fact that P gamma trypsin-activation and C-terminal truncation both lead to PDE activation. Furthermore, our findings on the mechanism of PDE inhibition of P gamma are relevant for understanding the mechanism of PDE activation by transducin.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

Dual coupling of opioid receptor-like (ORL1) receptors to adenylyl cyclase in the different layers of the rat main olfactory bulb.

The coupling of opioid receptor-like (ORL1) receptors to adenylyl cyclase has been investigated in specific layers of the rat main olfactory bulb. Membranes prepared from the olfactory nerve-glomerular layer (ON-G layer), external plexiform layer (EP layer) and granule cell layer (GR layer) displayed specific binding sites for [(3)H]-nociceptin/orphanin FQ ([(3)H]Noc/OFQ). In each layer, the presence of high-and low-affinity binding sites, with K(D) values in the picomolar and nanomolar range, respectively, was detected. The binding of [(3)H]Noc/OFQ was displaced by unlabelled Noc/OFQ, but not by opioid antagonists. In each layer, Noc/OFQ significantly stimulated [(35)S]GTPgammaS binding with nanomolar potencies. In ON-G layer, Noc/OFQ inhibited basal adenylyl cyclase activity and the enzyme stimulations by corticotropin releasing hormone (CRH), Ca(2+)/calmodulin (Ca(2+)/CaM) and forskolin (FSK). In EP layer, Noc/OFQ inhibited Ca(2+)/CaM-and FSK-stimulated enzyme activities. Conversely, in GR layer the peptide stimulated basal cyclase activity and potentiated the enzyme activation by CRH. The Noc/OFQ stimulation was counteracted by the GDP-bound form of the alpha subunit of transducin and was mimicked by transducin betagamma subunits. In the same tissue layer, Ca(2+)/CaM-and FSK-stimulated enzyme activities were inhibited. Naloxone failed to antagonize all the actions of Noc/OFQ. Western blot and RT-PCR analysis revealed the expression of Ca(2+)-insensitive and -sensitive adenylyl cyclases in the three layers. These results demonstrate that in rat main olfactory bulb ORL1 receptors can differentially affect distinct forms of adenylyl cyclase in a layer specific manner.

Adenylyl Cyclases↗
PubMed2005

Barx2 functions through distinct corepressor classes to regulate hair follicle remodeling.

The hair-growth cycle, a complex biological system requiring coordinate alterations in gene expression and cellular behavior, provides a challenging model for investigating the interplay of specific transcriptional regulation events. Here we report that the Barx2 homeodomain factor serves as a regulator of hair follicle remodeling (catagen), and loss of Barx2 in mice causes a defect both in the initiation and progression of catagen, resulting in a protracted first catagen, and later, causing short hair in adult gene-deleted mice. Barx2 negatively regulates its own promoter, and our study highlights the role of Barx2 as a repressor in the skin that can, unexpectedly, functionally interact with two WD40-domain factors distantly related to the yeast corepressor Tup1. These two corepressors, transducin-like enhancer of split and transducin beta-like 1, function through distinct and independent interactions with Barx2 for the repression of gene targets, including the Barx2 gene itself, emphasizing the roles of complementary repression strategies in engrailed homology-1 motif-containing homeodomain factors. Together, our data suggest that the hair-remodeling defect of Barx2 mutant mice could be explained, in part, by failure to repress one or more critical target genes.

Animals↗
PubMed1986

Epidermal growth factor (urogastrone)-mediated phosphorylation of a 35-kDa substrate in human placental membranes: relationship to the beta subunit of the guanine nucleotide regulatory complex.

We have identified a component of about 35 kDa (pp35), present in human placental membrane preparations, that is a substrate for epidermal growth factor (urogastrone) [EGF(Uro)]-mediated phosphorylation. The EGF(Uro)-stimulated phosphorylation of pp35 was calcium-dependent and was markedly enhanced in membranes prepared in the presence (but not in the absence) of calcium. The phosphate incorporated into pp35 in the presence of EGF(Uro) was alkali-stable and was present as O4-phosphotyrosine. Under identical conditions, insulin did not stimulate pp35 phosphorylation. Either in its native or in its phosphorylated form, pp35 could be released from the membranes in the presence of calcium-chelating agents (EDTA/EGTA); and EGF(Uro)-stimulated phosphorylation was reconstituted by adding back EDTA/EGTA eluates to EDTA/EGTA-washed membranes in the presence of calcium. The properties of pp35 were similar if not identical to those of beta-35, a 35-kDa polypeptide similar to the beta subunit of the guanine nucleotide-binding oligomers that stimulate (Gs) or inhibit (Gi) the adenylate cyclase system. As with pp35, EGF(Uro)-stimulated phosphorylation of isolated rabbit liver beta-35 was observed in a reconstituted system using either EDTA/EGTA-washed placental membranes or solubilized EGF(Uro) receptor immobilized on concanavalin A-agarose. In contrast, the addition of beta subunits derived from rabbit liver Gi or bovine transducin did not result in phosphorylation of a 35-kDa substrate in the reconstituted system. Further, a 35-kDa protein released from placental membranes crossreacted with an anti-transducin antibody that can recognize the beta subunit isolated from a variety of sources. We conclude that the human placental pp35 substrate likely represents the placental equivalent of the beta-35 protein. Our data point to a possible link between those receptors involved in growth-factor action and the regulatory systems that utilize GTP-binding proteins as transducing elements.

Calcium↗
PubMed1988

Specificity of action of guanine nucleotide-binding regulatory protein subunits on the cardiac muscarinic K+ channel.

The cardiac muscarinic receptor stimulates a potassium-selective ionic current (IK.ACh) through activation of a guanine nucleotide-binding regulatory protein. Purified alpha and beta gamma subunits of the guanine nucleotide-binding regulatory protein have each been reported to open the K+ channel. We have reported that nanomolar concentrations of purified brain beta gamma subunits activated IK.ACh in chicken embryonic atrial patches. In contrast, J. Codina, A. Yatani, D. Grenet, A.M. Brown, and L. Birnbaumer [(1987) Science 236, 442-445] subsequently reported that picomolar concentrations of activated erythrocyte alpha subunits (i.e., the 40-kDa alpha subunit that the authors call alpha K) opened K+ channels in guinea pig atrial patches. In this paper, we further explore the specificity of various beta gamma and alpha subunits in embryonic chicken and neonatal rat atrial patches. Beta gamma subunits from either human placenta (beta 35 gamma) or bovine brain (beta 35,36 gamma) activated IK.ACh whereas transducin beta gamma (beta 36 gamma) did not. The beta gamma activation was consistent in rat and chicken patches [118 of 123 patches (97%)]. Beta gamma subunits opened K+ channels at concentrations greater than or equal to 200 pM and maximally activated the channel at 10 nM. Beta gamma or guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) channel activation could be reversed by alpha 41-GDP. The purified brain beta gamma preparation was contaminated with less than 0.01% unactivated alpha. The detergent (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPS), used to suspend the hydrophobic beta gamma, did not activate IK.ACh alone, with buffer, with heat-inactivated beta gamma, or with transducin beta gamma. Unactivated alpha subunits did not open K+ channels. Activated, alpha subunits purified from human erythrocytes (alpha 40-GTP[gamma-S]) or bovine brain (alpha 39-GTP[gamma-S]) at concentrations of 10 pM or higher (up to 1 nM) opened K+ channels less frequently in chicken atrial patches [5 of 27 patches (19%) and 9 of 35 patches (26%), respectively] than in rat atrial patches [5 of 11 patches (45%) and 11 of 19 patches (58%), respectively]. Negative results were not due to patch vesicle formation. Other experiments indicated that alpha and beta gamma activated the same population of channels. Activation of the channel by both beta gamma and alpha subunits implies a more complicated scheme for guanine nucleotide-binding regulatory protein action than previously proposed.

Animals↗
PubMed1988

Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin.

Recent molecular cloning of cDNA for the alpha subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called Tr and Tc, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, we have identified a G-protein alpha subunit, which we refer to as Gz alpha, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine Tr alpha-subunit cDNA. The deduced amino acid sequence of Gz alpha is 41-67% identical with those of other known G-protein alpha subunits. However, the 355-residue Gz alpha lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein alpha subunits. We suggest that Gz alpha, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

Adenosine Diphosphate Ribose↗
PubMed1996

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

Amino Acid Sequence↗
PubMed1996

Responses of the phototransduction cascade to dim light.

The biochemistry of visual excitation is kinetically explored by measuring the activity of the cGMP phosphodiesterase (PDE) at light levels that activate only a few tens of rhodopsin molecules per rod. At 23 degrees C and in the presence of ATP, the pulse of PDE activity lasts 4 s (full width at half maximum). Complementing the rod outer segments (ROS) with rhodopsin kinase (RK) and arrestin or its splice variant p44 does not significantly shorten the pulse. But when the ROS are washed, the duration of the signal doubles. Adding either arrestin or p44 back to washed ROS approximately restores the pulse width to its initial value, with p44 being 10 times more efficient than arrestin. This supports the idea that, in vivo, capping of phosphorylated R* is mostly done by p44. When myristoylated (14:0) recoverin is added to unwashed ROS, the pulse duration and amplitude increase by about 50% if the free calcium is 500 nM. This effect increases further if the calcium is raised to 1 microM. Whenever R* deactivation is changed--when RK is exogenously enriched or when ATP is omitted from the buffer--there is no impact on the rising slope of the PDE pulse but only on its amplitude and duration. We explain this effect as due to the unequal competition between transducin and RK for R*. The kinetic model issued from this idea fits the data well, and its prediction that enrichment with transducin should lengthen the PDE pulse is successfully validated.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Molecular cloning and characterization of the G protein gamma subunit of cone photoreceptors.

The phototransduction process in cones has been proposed to involve a G protein that couples the signal from light-activated visual pigment to the effector cyclic GMP phosphodiesterase. Previously, we have identified and purified a G beta gamma complex composed of a G beta 3 isoform and an immunochemically distinct G gamma subunit (G gamma 8) from bovine retinal cones (Fung, B. K.-K., Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267, 24782-24788; Lee, R. H., Lieberman, B.S., Yamane, H. K., Bok, D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267, 24776-24781). Based on the partial amino acid sequence of this cone G gamma 8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding G gamma 8. The cDNA insert of this clone includes an open reading frame of 207 bases encoding a 69-amino acid protein. The predicted protein sequence of G gamma 8 shares a high degree of sequence identity (68%) with the G gamma (G gamma 1) subunit of rod transducin. Similar to rod G gamma 1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for G gamma 8 is present at a high level in the retina and at a very low level in the lung, but is undetectable in other tissues. Immunostaining of bovine retinal sections with an antipeptide antibody against the N-terminal region of G gamma 8 further shows a differential localization of G gamma 8 to cones with a pattern indistinguishable from that of G beta 3. This finding suggests that G beta 3 gamma 8 is a component of cone transducin involved in cone phototransduction and color vision.

Amino Acid Sequence↗
PubMed1996

Interactions of phosducin with defined G protein beta gamma-subunits.

Phosducin has recently been identified as a cytosolic protein that interacts with the beta gamma-subunits of G proteins and thereby may regulate transmembrane signaling. It is expressed predominantly in the retina but also in many other tissues, which raises the question of its potential specificity for retinal versus nonretinal beta gamma-subunits. We have therefore expressed and purified different combinations of beta- and gamma-subunits from Sf9 cells and have also purified transducin-beta gamma from bovine retina and a mixture of beta gamma complexes from bovine brain. Their interactions with phosducin were determined in a variety of assays for beta gamma function: support of ADP-ribosylation of alpha 0 by pertussis toxin, enhancement of the GTPase activity of alpha 0, and enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase 1 (betaARK1). There were only moderate differences in the effects of the various beta gamma complexes alone on alpha 0, but there were marked differences in their ability to support betaARK1 catalyzed rhodopsin phosphorylation. Phosducin inhibited all beta gamma-mediated effects and showed little specificity toward specific defined beta gamma complexes with the exception of transducin-beta gamma (beta1 gamma1), which was inhibited more efficiently than the other beta gamma combinations. In a direct binding assay, there was no apparent selectivity of phosducin for any beta gamma combination tested. Thus, in contrast to betaARK1, phosducin does not appear to discriminate strongly between different G protein beta- and gamma-subunits.

Adenosine Diphosphate Ribose↗
PubMed1996

Presence of a light-independent phospholipase A2 in bovine retina but not in rod outer segments.

Rod outer segments (ROS) are responsible for the visual transduction process. Rhodopsin, which constitutes 85-90% of ROS proteins, absorbs light photons, changes its conformation, and then binds to a heterotrimeric G-protein called transducin. As a consequence, transducin dissociates into Talpha and Tbetagamma subunits. The presence in ROS of a phospholipase A2 (PLA2) stimulated by light and guanosine 5'-O-(3-thio)triphosphate was first demonstrated in 1987 (Jelsema, C. L.(1987) J. Biol. Chem. 262, 163-168). This led that author to conclude that ROS PLA2 could be involved in the phototransduction process, and raised the possibility of receptor-mediated activation of PLA2 via G-proteins in cell types other than rods. However, the biochemical characteristics and the role of this PLA2 have not been fully elucidated. We have tried to reproduce some of the results previously reported in order to further characterize this enzyme. We have found that, in our hands, there is neither light-dependent nor GTP-dependent PLA2 activity in intact purified ROS. We also failed to detect PLA1 activity in those ROS preparations. Nevertheless, we detected significant amounts of PLA2 activity in two subretinal fractions adjacent to ROS: RPE (enriched with retinal pigment epithelial cells) and P200 (presumably containing neuronal cells, Müller cells, and rod inner segments). The enzyme present both in RPE and P200 is light- and GTP-independent, Ca2+- and Mg2+-independent, and seems to be optimally active in the alkaline pH range. Our results suggest that there is, if any, vanishingly little PLA2 or PLA1 activity in intact purified ROS and that the activity levels previously reported in the literature could have been due to a contamination by either RPE or P200. This is supported by our observation that some contaminated ROS preparations were "PLA2 active."

Animals↗
PubMed1996

An interface of interaction between photoreceptor cGMP phosphodiesterase catalytic subunits and inhibitory gamma subunits.

Cyclic guanosine 5'-monophosphate (cGMP) phosphodiesterase (PDE) regulates the level of cGMP on transduction of a visual signal in vertebrate photoreceptor cells. Two identical inhibitory PDE gamma subunits (Pgammas) block catalytic activity of PDE-alpha and -beta subunits (Palphabeta) in the dark. The primary regions of Pgamma involved in the interaction with Palphabeta are a central polycationic region, Pgamma-24-45, and a C-terminal region of Pgamma. Recently, we have shown that the C-terminal region of Pgamma, which is the major Pgamma inhibitory domain, blocks PDE activity by binding to the catalytic site of PDE (Artemyev, N. O., Natochin, M., Busman, M., Schey, K. L., and Hamm, H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412). Here, we localize the site on the rod cGMP PDE alpha subunit that binds to the central polycationic domain of Pgamma. This site is located within a region that links a second noncatalytic cGMP binding site with the catalytic domain of PDE. A polypeptide coresponding to this region, Palpha-461-553, expressed as a glutathione S-transferase fusion protein in Escherichia coli and isolated after cleavage of the fusion protein with thrombin, blocks inhibition of PDE activity by Pgamma. In addition, Palpha-461-553 binds to the Pgamma-24-45 region (Kd, 7 microM), as measured by a fluorescent increase in a Pgamma-24-45Cys peptide labeled with 3-(bromoacetyl)-7-diethylaminocoumarin. The Palpha-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of Palpha (Palpha-517-541) effectively suppressed inhibition of PDE activity by Pgamma and bound to Pgamma-24-45Cys labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Kd, 22 microM). Palpha-517-541 also competes with the activated rod G-protein alpha-subunit for binding to Pgamma labeled with lucifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transducin alpha-guanosine 5'-triphosphate and Palpha-517-541 for binding to the Pgamma-24-45 region. Based on the results, we propose a linear model of interactions between catalytic and inhibitory PDE subunits.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

Prolonged activation of extracellular signal-regulated kinase by a protein kinase C-dependent and N17Ras-insensitive mechanism mediates the proliferative response of G(i/o)-coupled somatostatin sst(4) receptors.

The human sst(4) receptor, recombinantly expressed in Chinese hamster ovary cells, mediates proliferative activity of the peptide hormone somatostatin. This effect was shown to involve activation of pertussis toxin-sensitive G proteins and was inhibited by overexpression of the betagamma-sequestrant, transducin. Somatostatin-induced proliferation was abolished by the MEK1 inhibitor, PD 98059, whereas the Src inhibitor, PP1, had no effect. A marked increase was observed in the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) 10 min after sst(4) receptor activation, which was blocked by pertussis toxin, decreased by PP1 and the betagamma-sequestrant, but unaffected by PD 98059. In contrast, the somatostatin-induced phosphorylation of ERK obtained at 4 h, although sensitive to both pertussis toxin and transducin, was unaffected by PP1 but ablated by PD 98059. Protein kinase C inhibition also abolished this somatostatin-induced sustained phosphorylation of ERK, together with the associated increase in cell proliferation. Expression of dominant negative Ras (N17) failed to significantly reduce the proliferative effect mediated by the sst(4) receptor but markedly attenuated the acute phase of the somatostatin-induced phosphorylation of ERK obtained at 10 min. In contrast, the phosphorylation induced at 4 h was unaffected. We conclude that ERK activation by G(i/o)-coupled sst(4) receptors involves a Src and Ras-dependent acute phase, but the proliferative response is dependent upon the prolonged ERK-induced activity, mediated by protein kinase C.

Animals↗
PubMed2000

Enhancement of phototransduction protein interactions by lipid surfaces.

The G protein cascade of vision depends on two peripheral membrane proteins: the G protein, transducin (G(t)), and cGMP phosphodiesterase (PDE). Each has covalently attached lipids, and interacts with transduction components on the membrane surface. We have found that their surface interactions are critically dependent on the nature of the lipid. Membranes enhance their protein-protein interactions, especially if electrostatic attraction is introduced with positively charged lipids. These interactions are less enhanced on highly curved surfaces, but are most enhanced by unsaturated or bulky acyl chains. On positively charged membranes, G(t) assembles at a high enough density to form two-dimensional arrays with short-range crystalline order. Cationic membranes also support extremely efficient activation of PDE by the GTPgammaS (guanosine 5'-O-(thiotriphosphate)) form of Galpha(t) (Galpha(t)-GTPgammaS), minimizing functional heterogeneity of transducin and allowing activation with nanomolar Galpha(t)-GTPgammaS. Quantification of PDE activation and of the amount of Galpha(t)-GTPgammaS bound to PDE indicated that G(t) activates PDE maximally when bound in a 1:1 molar ratio. No cooperativity was observed, even at nanomolar concentrations. Thus, under these conditions, the one binding site for Galpha(t)-GTPgammaS on PDE that stimulates catalysis must be of higher affinity than one or more additional sites which are silent with respect to activation of PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5.

Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2002

Specific binding of RGS9-Gbeta 5L to protein anchor in photoreceptor membranes greatly enhances its catalytic activity.

The complex between the short splice variant of the ninth member of the RGS protein family and the long splice variant of type 5 G protein beta subunit (RGS9-Gbeta5L) plays a critical role in regulating the duration of the light response in vertebrate photoreceptors by activating the GTPase activity of the photoreceptor-specific G protein, transducin. RGS9-Gbeta5L is tightly associated with the membranes of photoreceptor outer segments; however, the nature of this association remains unknown. Here we demonstrate that rod outer segment membranes contain a limited number of sites for high affinity RGS9-Gbeta5L binding, which are highly sensitive to proteolysis. In membranes isolated from bovine rod outer segments, all of these sites are occupied by the endogenous RGS9-Gbeta5L, which prevents the binding of exogenous recombinant RGS9-Gbeta5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gbeta5L, allowing for high affinity binding of recombinant RGS9-Gbeta5L to these sites. This binding results in a striking approximately 70-fold increase in the RGS9-Gbeta5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gbeta5L membrane attachment, as evident from the analysis of membrane-binding properties of deletion mutants lacking either N- or C-terminal parts of the RGS9 molecule. Our data indicate that specific association of RGS9-Gbeta5L with photoreceptor disc membranes serves not only as a means of targeting it to an appropriate subcellular compartment but also serves as an important determinant of its catalytic activity.

Amino Acid Substitution↗
PubMed2002

Rhodopsin with 11-cis-locked chromophore is capable of forming an active state photoproduct.

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C(11)=C(12) double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C(11)=C(12) double bond is prevented by a 6-member ring structure (Rh(6.10)). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C(11)=C(12) double bond being locked, isomerization around the C(9)=C(10) or the C(13)=C(14) double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.

Hydrogen-Ion Concentration↗
PubMed2004

Tax deregulation of NF-kappaB2 p100 processing involves both beta-TrCP-dependent and -independent mechanisms.

Processing of the nf-kappab2 gene product p100 to generate p52 is a tightly regulated event, consistent with the fact that the processing product, p52, is hardly detected in most cell types, including T cells, although the precursor p100 is expressed abundantly in these cells. However, in T cells transformed by the human T-cell leukemia virus type I (HTLV-I), p100 processing is very active, resulting in high level expression of p52. Because overproduction of p52 is associated with lymphoid hyperplasia and transformation, deregulation of p100 processing may be part of the oncogenic mechanism of HTLV-I. We demonstrated previously that HTLV-I Tax oncoprotein is a potent inducer of p100 processing through specific targeting of IKKalpha via IKKgamma to p100 to trigger p100 phosphorylation and ubiquitination. In this study, we further show that Tax-mediated recruitment of IKKalpha to p100 requires serines 866 and 870 of p100, shown to be essential for inducible processing of p100. Upon interaction with p100, activated IKKalpha phosphorylates both N- and C-terminal serines of p100 (serines 99, 108, 115, 123 and 872), serving as a critical step in Tax-induced p100 processing. Using a genetic approach, we find that beta-transducin repeat-containing protein, a component of the SCF ubiquitin ligase complex, previously shown to be required for physiological p100 processing mediated by nuclear factor-kappaB-inducing kinase, is only partially involved in Tax-induced processing of p100. These results indicate that both beta-transducin repeat-containing protein-dependent and -independent mechanisms contribute to Tax-deregulated p100 processing, further suggesting the involvement of different mechanisms in cellular and viral pathways of p100 processing.

Cell Line, Transformed↗