Sulfhydryl group modification of photoreceptor G-protein prevents its light-induced binding to rhodopsin

The effect of sulfhydryl modification on the light-induced interaction between rhodopsin and the peripheral GTP-binding protein of the photoreceptor membrane (G-protein) has been investigated by time-resolved near-infrared light-scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N-ethylmaleimide (NEM) does not affect its light-induced interaction with the G-protein. Modification of G-protein with NEM or other sulfhydryl agents prevents any light-induced binding to rhodopsin. Dark-association of G to the membrane as well as the light-induced complex with rhodopsin (once formed) is insensitive to NEM.     

Squid visual arrestin: cDNA cloning and calcium-dependent phosphorylation by rhodopsin kinase (SQRK)

Arrestin binding to rhodopsin is one of the major mechanisms of termination of photoresponses in both vertebrates and invertebrates. Here we report the cDNA cloning and characterization of a 48-kDa visual arrestin from squid (Loligo pealei). The cDNA encoded a protein that had 56-64% amino acid sequence similarity to reported arrestin sequences. This protein does not encode any distinct modular domains but contains five fingerprint regions that have been identified within arrestins. Antibodies raised to the recombinant arrestin protein detected arrestin expression only in the eye and recognized a doublet in photoreceptor membranes, representing unphosphorylated and phosphorylated arrestin. In squid eye membranes, arrestin was phosphorylated in a Ca2+-dependent manner and this phosphorylation was inhibited by antibodies raised against squid rhodopsin kinase, but not by inhibitors of protein kinase C or calmodulin kinase. Addition of purified squid rhodopsin kinase to washed rhabdomeric membranes resulted in phosphorylation of rhodopsin, and arrestin was also phosphorylated when calcium was present. This is the first report of a rhodopsin kinase phosphorylating an arrestin substrate, and suggests a dual role for this kinase in the inactivation of the squid visual system.     

Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

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.     

A light-stimulated increase of cyclic GMP in squid photoreceptors

Photoreceptor outer segments isolated from squid retina are known to contain a light-activated GTP-binding protein. Here it is shown that these photoreceptors contain around 0.01 mol cyclic GMP per mol rhodopsin. Adding GTP in the dark stimulates the production of 0.0003-0.001 mol cyclic GMP/mol rhodopsin per min. GTP and light cause a 2-fold faster increase in cyclic GMP. These results show that either (1) squid rhodopsin activates a guanylate cyclase, or (2) there is a constant guanylate cyclase activity and photoexcited rhodopsin inhibits a cyclic GMP phosphodiesterase.     

Eye diseases and proteins controlling visual transduction

Retinal S antigen is a soluble protein found in abundance in photoreceptor cells. Immunization of laboratory animals with this antigen in adjuvant induces experimental autoimmune uveoretinitis. Cellular immunity plays a major role in this condition. Autoimmune responses toward retinal S antigen are often observed in patients with retinal inflammatory disease, however, these responses are usually secondary to local tissue damage. The S antigen is identical to the 48 K protein characterized in rod outer segments by its light-dependent binding to the disk membrane in the presence of ATP. This protein binds specifically to photoexcited and phosphorylated rhodopsin, and quenches the activity of the light-dependent cGMP-phosphodiesterase, most probably because it competes with transducin. There is no evidence for any direct inactivation of phosphodiesterase by 48 K protein. In view of the numerous similarities between the photoreceptor enzyme cascade and hormone-activated cyclase systems, a related protein could be involved in the desensitization of hormonal systems.     

Light-modulated ADP-ribosylation, protein phosphorylation and protein binding in isolated fly photoreceptor membranes

Rhodopsin (P, lambda max 480 nm) of blowfly photoreceptors R1-6 is converted by light into a thermally stable metarhodopsin (M, lambda max 565 nm). In isolated blowfly rhabdoms photoconversion of P to M affects bacterial toxin-catalyzed ADP-ribosylation of a 41-kDa protein, activates phosphorylation of opsin and induces the binding of a 48-kDa phosphoprotein to the rhabdomeric membrane. ADP-ribosylation of the 41-kDa protein is catalyzed by cholera toxin and is inhibited by P----M conversion. The 41-kDa protein might represent the alpha-subunit of the G-protein, proposed to be part of the phototransduction mechanism [Blumenfeld, A. et al. (1985) Proc. Natl Acad. Sci. USA 82, 7116-7120]. P----M conversion leads to phosphorylation of opsin at multiple binding sites: up to 4 mol phosphate are bound/mol M formed. Dephosphorylation of the phosphate binding sites is induced by photoconversion of M to P. High levels of calcium (2 mM) inhibit phosphorylation of M and increase dephosphorylation of P. Protein patterns obtained by sodium dodecyl sulfate gel electrophoresis of irradiated retina membranes show an increased incorporation of label from [gamma-32P]ATP also into protein bands of 48 kDa, 68 kDa and 200 kDa. Binding studies reveal that in the case of the 48-kDa protein this effect is primarily due to a light-induced binding of the protein to the photoreceptor membrane. The binding of the 48-kDa phosphoprotein is reversible: after M----P conversion the protein becomes extractable by isotonic buffers. These data suggest that in rhabdomeric photoreceptors of invertebrates light-activation of rhodopsin is coupled to an enzyme cascade in a similar way as in the ciliary photoreceptors of vertebrates, although there may be differences, e.g. in the type of G-protein which mediates between the activated state of metarhodopsin and a signal-amplifying enzyme reaction.     

Study of the Interaction of the Myelin Monomeric GTP-Binding Proteins with Other Brain Proteins

Abstract: Although several monomeric GTP-binding proteins have been found in myelin, the signaling pathways in which they operate are not known. To define these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP-binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The presence of proteins that interact with the myelin GTP-binding proteins was explored by incubating those blots with an enriched fraction of 22- and 25-kDa myelin GTP-binding proteins labeled with radioactive guanine nucleotides. When the GTP-binding proteins were in the inactive state (GDP-bound) they interacted with 28-, 47-, and 58-kDa oligodendrocyte polypeptides. Only the 28-kDa protein was present in myelin. In the active state (GTP-bound), they interacted only with a 47-kDa protein in myelin but with 31-, 38-, 47-, 58-, 60-, 68-, and 71-kDa proteins in oligodendrocytes and total homogenate. Under these experimental conditions the 28-kDa protein did not interact with the GTP-binding proteins. The fact that the myelin GTP-binding proteins in the active state formed complexes with a different set of proteins than when in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31- and 38-kDa proteins that were detected only in the cytoplasmic fraction, these polypeptides were detected in the cytosolic fraction and total membrane fraction. The 25-kDa GTP-binding protein was present in all the complexes. Immunoblot analysis indicated that the 28-kDa polypeptide is RhoGDI, an effector protein that is known to regulate the activation and movement of several GTP-binding proteins between different cellular compartments. Thus, this study opens the way to identify the macromolecules participating in the myelin signaling pathway involving monomeric GTP-binding proteins.     

The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction.

The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.     

Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II

We have found that the 48-kDa protein (or S-antigen 48k) of the rod photoreceptor enhances the light-induced formation of the photoproduct metarhodopsin II (MII) from prephosphorylated rhodopsin. The effect is analogous to the known enhancement of MII (extra-MII) that results from selective interaction of MII with G-protein. We have determined some parameters of the MII-48k interaction by measuring the extra-MII absorption change induced by the 48-kDa protein. The amplitude saturation yields a dissociation constant for the MII-48k complex on the order of 50 nM. At the technical limit of these measurements, 13.7 degrees C and 12 microM 48-kDa protein, we find a rate of 2.3 s-1 for formation of the 48k-MII complex. Extrapolation of these values to cellular conditions yields an occupation time of phosphorylated MII by 48k less than 200 ms. This is short compared to estimated rates of phosphorylation. The temperature dependence of the MII-48k formation rate is very high (Q10 for 5 degrees C/15 degrees C = 9-10). The related Arrhenius activation energy (165 kJ mol-1) is correspondingly high and indicates a considerable transient chemical change during the binding process.     

Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation

In the vertebrate rod outer segment (ROS), the light-dependent activation of a GTP-binding protein (G-protein) and phosphodiesterase (PDE) is quenched by a process that requires ATP [Liebman, P.A., & Pugh, E.N. (1979) Vision Res. 19, 375-380]. The ATP-dependent quenching mechanism apparently requires the phosphorylation of photoactivated rhodopsin (Rho*); however, a 48-kilodalton protein (48K protein) has also been proposed to participate in the inactivation process. Purified species of phosphorylated rhodopsin containing 0, 2, or greater than or equal to 4 (high) phosphates per rhodopsin (PO4/Rho) were reconstituted into phosphatidylcholine (PC) vesicles and reassociated with a hypotonic extract from isotonically washed disk membranes that were depleted of 48K protein; PDE activation, in response to bleaching from 0.01% to 15% of the rhodopsin present, was measured. PDE activity was reduced by at least 30% at high fractional rhodopsin bleaches and by greater than 80% at low fractional rhodopsin bleaches in high PO4/Rho samples when compared to the activity measured in O PO4/Rho controls. A phosphorylation level of 2 PO4/Rho produced PDE activities that were intermediate between O PO4/Rho and high PO4/Rho samples at low bleaches, but were identical with the O PO4/Rho samples at high rhodopsin bleaches. Rhodopsin phosphorylation is thus capable of producing a graded inhibition of light-stimulated PDE activation over a limited range of (near physiological) bleach levels. This effect become less pronounced as the bleach levels approach those that saturate PDE activation. These results are consistent with increasing levels of phosphorylation, producing a reduction of the binding affinity of G-protein for Rho*.     

Interaction between photoexcited rhodopsin and peripheral enzymes in frog retinal rods. Influence on the postmetarhodopsin II decay and phosphorylation rate of rhodopsin

The major peripheral and soluble proteins in frog rod outer segment preparations, and their interactions with photoexcited rhodopsin, have been compared to those in cattle rod outer segments and found to be similar in both systems. In particular the GTP-binding protein (G) has the same subunit composition, the same abundance relative to rhodopsin (1/10) and it undergoes the same light and nucleotide-dependent interactions with rhodopsin in both preparations. Previous work on cattle rod outer segments has shown that photoexcited rhodopsin (R*), in a state identified with metarhodopsin II, associates with the G protein as a first step to the light-activated GDP/GTP exchange on G. The complex R*-G is stable in absence of GTP, but is rapidly dissociated by GTP owing to the GDP/GTP exchange reaction. Low bleaching extents (less than 10% R*) in absence of GTP therefore create predominantly R*-G complexes, whereas bleaching in presence of GTP creates free R*. We report here that, under conditions of complexed R*, two reactions of R* in frog rod outer segments are highly perturbed as compared to free R*: (a) the spectral decay of metarhodopsin II (MII) into later photoproducts, and (b) the phosphorylation of R* by an ATP-dependent protein kinase. a) The spectral measurements have been performed using linear dichroism on oriented frog rod outer segments; this technique allows discrimination between MII and later photoproducts absorbing at the same wavelength. Association of R* with G leads to a strong reduction of the amount of MIII formed and to an acceleration of the decay of MIII. Furthermore, MII is significantly stabilized, in agreement with the hypothesis that MII is the intermediate which binds to G. b) The phosphorylation of R* is strongly inhibited under conditions of R*-G complex formation as compared to free R*. Interferences between reactions at the three sites involved in R* are discussed: the retinal binding site in the hydrophobic core is sensitive to the presence of GTP-binding protein at its binding site on the cytoplasmic surface of R*; the kinase and the GTP-binding protein compete for access to their respective binding sites, both located on the surface of R*. We also observed a slow and nucleotide-dependent light-induced binding of a protein of molecular weight 50 000, which we consider as the equivalent of the 48 000 Mr light-dependent protein previously identified in cattle rod outer segments.     

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.     

Regulation of rhodopsin dephosphorylation by arrestin

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.     

Direct binding of visual arrestin to microtubules determines the differential subcellular localization of its splice variants in rod photoreceptors

Proper function of visual arrestin is indispensable for rapid signal shut-off in rod photoreceptors. Dramatic light-dependent changes in its subcellular localization are believed to play an important role in light adaptation of photoreceptor cells. Here we show that visual arrestin binds microtubules. The truncated splice variant of visual arrestin, p44, demonstrates dramatically higher affinity for microtubules than the full-length protein (p48). Enhanced microtubule binding of p44 underlies its earlier reported preferential localization to detergent-resistant membranes, where it is anchored via membrane-associated microtubules in a rhodopsin-independent fashion. Experiments with purified proteins demonstrate that arrestin interaction with microtubules is direct and does not require any additional protein partners. Most importantly, arrestin interactions with microtubules and light-activated phosphorylated rhodopsin are mutually exclusive, suggesting that microtubule interaction may play a role in keeping p44 arrestin away from rhodopsin in dark-adapted photoreceptors.     

Properties of a blue-light-absorbing photoreceptor kinase localized in the plasma membrane of the coleoptile tip region

The blue-light-sensing apical part of coleoptiles of grasses is responsible for the first positive phototropic bending reaction of this organ. The photoreceptor responsible has been shown to be localized to the plasma membrane (PM) of this tip region. An approximately 100-kDa protein moiety of this receptor is rapidly phosphorylated upon irradiation. Properties of this protein kinase reaction were studied in vitro by using PMs from the maize (Zea mays L.) coleoptile tip region: (i) The substrate for the blue-light-triggered phosphorylation of the 100-kDa protein was found to be ATP as well as GTP. However, the affinity of the involved protein kinase for the substrate GTP was lower than for ATP. (ii) Experiments were undertaken to find out whether a photoreceptor moiety acts as an autophosphorylating protein kinase or whether the photoreceptor protein, when activated by light, becomes the target of an extrinsic protein kinase. Two studied extrinsic protein kinases (50 and 55 kDa) of the coleoptile tip were found not to be involved in the lightdependent protein phosphorylation. The degree of phosphorylation of the 100-kDa protein on isolated plasma membranes upon irradiation at 0 °C was scarcely different from a reaction at 30 °C, in contrast to the background protein phosphorylations which decreased with decreasing temperature. This result points to an autophosphorylation mechanism at the receptor. (iii) In mixing experiments, solubilized membranes from maize coleoptiles were irradiated and added to unirradiated membrane proteins from pea (Pisum sativum L.) epicotyls followed by addition of [-³²P]ATP. Unirradiated proteins from pea were not phosphorylated by light-activated (autophosphorylatable) maize protein kinases. (iv) It is suggested that the blue-light-sensitive photoreceptor localized to the PM of the phototropically active tip region of coleoptiles has an autophosphorylatable kinase domain which is able to use ATP or GTP as substrate.Abbreviation PM plasma membrane The author is deeply indebted to Mrs Elke Stransky for her excellent technical assistance and performance of the experiments.     

Purification and characterization of a probable light receptor with kinase activity from beet root plasma membranes

A 120-kDa glycoprotein was found in beet root (Beta vulgaris L.) plasma membranes. This protein could be phosphorylated in a Ca2+-independent manner. Its carbohydrate moiety was composed of both O-linked galactose-beta(1-3)-N-acetylgalactosamine disaccharides (which bind peanut agglutinin) and N-linked concanavalin A (ConA)-binding oligosaccharides. The phosphorylation of this protein was rapid, half-saturated with 6 microM ATP and higher at alkaline pH values. This protein was phosphorylated more efficiently with Mn-ATP as substrate than with Mg-ATP. This phosphorylation increased when plasma membranes were illuminated with low-fluence blue light, a fact suggesting that the 120-kDa glycoprotein could be similar to phototropin: a blue-light photoreceptor involved in phototropism. This protein was purified using a ConA-Sepharose column. The phosphorylation of the purified protein could be observed, but it was much lower than that of the 120-kDa protein in plasma membranes. In addition, it was not enhanced by light. Some possible explanations for this photosensitivity loss are discussed.     

A variant of arrestin-1 binds rod outer segment membranes in a light-independent manner

A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule. However, in contrast to arrestin-1 that associated to the ROS membranes only in the presence of light and ATP, the 50-kDa protein interacted with the ROS membranes in a light-independent manner, either in the presence or absence of ATP. These results clearly established that phosphorylated and illuminated rhodopsin is not the membrane anchor for this variant of arrestin-1.     

Pollination-induced senescence in phalaenopsis petals. Relationship of ethylene sensitivity to activity of GTP-binding proteins and protein phosphorylation

Treatments of cut phalaenopsis ( Phalaenopsis hybrid , cv. 'Herbert Hager') flowers with cholera toxin or guanosine-5-0-(3-thiotriphosphate), compounds that modulate GTP-binding protein activity, increased the sensitivity of the flowers to ethylene. Guanosine-5-0-(2-thiodiphosphate) which does not affect the activity of GTP-binding proteins, had no affect on the sensitivity to ethylene. Western blot analysis of microsomal proteins, revealed that a peptide with a molecular mass of ca 42 kDa cross-reacts with antibodies against a well-conserved amino acid sequence (G_α-commun peptide) of mammalian G-proteins. Calcium ions, known co-factors of protein kinases, also increased the sensitivity of the flowers to ethylene, while EGTA, a chelator of calcium, decreased it. Phorbol 12-myrisate 13-acetate, a phorbol ester, had no effect on the sensitivity to ethylene. Protein phosphorylation in petal microsomal membranes was doubled in the presence of calcium ions, but was unaffected by phorbol ester. Ten h after pollination, at the peak of ethylene sensitivity, a significant increase of ca 20% was measured in the binding of GTP to the membranes. Protein phosphorylation in flowers increased significantly following pollination, with a single peptide of ca 30 kDa most heavily phosphorylated. These observations may indicate a direct involvement of GTP-binding proteins, and protein phosphorylation, two major components of the cellular signal transduction pathway, in the regulation of pollination induced ethylene sensitivity in phalaenopsis petals.     

Uncoupling of Rat Cerebral Cortical α2-Adrenoceptors from GTP-Binding Proteins by N-Ethylmaleimide

Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.