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H E Hamm

Publications and source records attributed to H E Hamm.

At least 19 recordsLinked to original sources
PubMed1995

An effector site that stimulates G-protein GTPase in photoreceptors.

Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the alpha-subunit with GTP. alpha-Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase gamma-subunits (PDE gamma) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysis by transducin in a complex with this mutant. This mutation also resulted in a reduction of PDE gamma affinity for transducin, but did not affect PDE gamma interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE gamma to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDE gamma in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.

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

The carboxyl terminus of the gamma-subunit of rod cGMP phosphodiesterase contains distinct sites of interaction with the enzyme catalytic subunits and the alpha-subunit of transducin.

The interaction between the GTP-bound form of the transducin alpha-subunit (G alpha t) and the gamma-subunit (P gamma) of cGMP phosphodiesterase (PDE) is a key event in effector activation during photon signal transduction. The carboxyl-terminal half of P gamma is involved in interaction with G alpha t as well as in inhibition of PDE activity. Here we have utilized a combination of synthetic peptide and mutagenesis approaches to localize specific regions of the carboxyl-terminal region of P gamma interacting with G alpha t and P alpha beta and have determined residues involved in inhibition of PDE activity. We found that synthetic peptide corresponding to residues 68-87 of P gamma completely inhibit trypsin-activated PDE. The peptide P gamma-63-87 bound to G alpha t GTP gamma S with a Kd of 2.5 microM, whereas the binding of P gamma-68-87 to G alpha tGTP gamma S was approximately 15-fold less (Kd = 40 microM) suggesting that carboxyl-terminal P gamma region 68-87 contains a site for interaction with P alpha beta and also a part of the alpha t binding site. To map G alpha t and P alpha beta sites more precisely within the carboxyl-terminal region, a set of carboxyl-terminal mutants was generated by site-directed mutagenesis. Deletion of residues 63-69 and 70-76 diminished the binding of mutants to alpha t while binding to carboxyl-terminally truncated mutants lacking up to 11 amino acid residues was unchanged. In contrast, carboxyl-terminal truncations of P gamma from delta 1 to delta 11 resulted in a gradual decrease of its inhibitory activity. Thus, the extreme carboxyl-terminal hydrophobic sequence -Ile86-Ile87 together with 9 adjacent residues provides inhibitory interaction of P gamma with P alpha beta. The carboxyl-terminal G alpha tGTP gamma S binding site of P gamma is different from but adjacent to its PDE inhibitory site. During the visual transduction process, G alpha tGTP likely binds to this region of P gamma inducing a displacement of the extreme carboxyl terminus from the inhibitory site on P alpha beta, leading to PDE activation.

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

Structural and functional relationships of heterotrimeric G-proteins.

Heterotrimeric GTP-binding proteins (G-proteins) are a critical component of signal transduction pathways that carry information received at the cell surface to the appropriate cellular effector system, ultimately achieving a specific cellular response. Heterotrimeric G-proteins consist of an alpha-subunit, which contains the guanine nucleotide binding site and intrinsic GTPase activity, and an inseparable beta gamma-subunit complex. G-proteins act to define the specificity by which a receptor regulates a particular intracellular signaling system, as well as to regulate the duration of the signal. A great deal of structural and functional insight into how G-protein-mediated signal transduction occurs has recently been achieved. This review will discuss the structural features of G-proteins, as well as detail the mechanism by which G-proteins interact with receptors and effectors.

GTP-Binding Proteins↗
PubMed1994

GTPase mechanism of Gproteins from the 1.7-A crystal structure of transducin alpha-GDP-AIF-4.

Aluminium fluoride (AIF-4) activates members of the heterotrimeric G-protein (G alpha beta gamma) family by binding to inactive G alpha.GDP near the site occupied by the gamma-phosphate in G alpha.GTP (ref. 3). Here we describe the crystal structure of transducin alpha.GDP activated with aluminium fluoride (Gt alpha.GDP.AIF-4.H2O) at 1.7 A, a resolution sufficient to establish the coordination geometry of the bound aluminium fluoride as well as the extensive network of direct and water-mediated interactions that stabilize it. These observations are derived from three independent representations in the asymmetric unit, eliminating any chance of drawing conclusions based on stereochemistry imposed by crystal packing. Surprisingly, aluminium fluoride activates G alpha.GDP by binding with a geometry resembling a pentavalent intermediate for GTP hydrolysis. The stabilizing interactions involve not only residues that interact with the gamma-phosphate in Gt alpha.GTP gamma S, but also conserved residues for GTPase activity. Thus the Gt alpha.GDP.AIF-4.H2O structure provides new insight into the mechanism of GTP hydrolysis.

Aluminum Compounds↗
PubMed1994

Synthetic peptides as probes for G protein function. Carboxyl-terminal G alpha s peptides mimic Gs and evoke high affinity agonist binding to beta-adrenergic receptors.

The molecular interfaces between Gs and the beta-adrenergic receptor were investigated using synthetic peptides corresponding to various regions of its alpha subunit, alpha s. These experiments were carried out on saponin-permeable C6 glioma cells in which the beta-adrenergic receptor appears tightly coupled to Gs. Synthetic site-specific peptides from alpha s (corresponding to amino acids 15-29, 354-372, and 384-394) and alpha i (8-22, 315-324, and 345-455) were tested for their ability to interfere with coupling between the beta-adrenergic receptor and Gs. The two carboxyl-terminal peptides from alpha s blocked beta-adrenergic stimulation of adenylyl cyclase in permeable cells. However, only alpha s-354-372 had this effect in C6 membranes. It is suggested that the partial uncoupling of Gs, which occurs subsequent to cell disruption, may be related to a change in the interaction of the alpha s carboxyl terminus with the beta-adrenoreceptor. Two carboxyl-terminal peptides, 354-372 and 384-394, could also mimic the effect of Gs to increase agonist affinity for the beta-adrenergic receptor. In combination, alpha s-354-372 and alpha s-384-394 increased the ability of isoproterenol to compete with 125I-pindolol binding in a partially additive manner. Synthetic peptides from alpha i and amino-terminal peptides from alpha s had no effect on beta-agonist binding, suggesting a high specificity of peptide effects. Two findings suggest that these peptides bind directly to the beta-adrenergic receptor and stabilize its high agonist affinity conformation. First, GTP and hydrolysis-resistant GTP analogs did not alter the high affinity binding in the presence of high concentrations of the peptides. Second, in S49 lymphoma cyc- cells, which lack Gs, these peptides evoked the high affinity agonist binding state of the beta-receptor. Neither peptide had an effect on antagonist binding affinity, as measured by propranolol displacement of 125I-pindolol. These data suggest that at least two regions on the alpha subunit of Gs participate in high affinity Gs binding to the beta-adrenergic receptor. The fact that these small peptides could mimic the holo-Gs effect on the receptor is rather surprising, and the specificity of the effect suggests that the primary and secondary structure of small regions of alpha s contain much of the information for specific interaction with beta-adrenergic receptors.

Adenylyl Cyclases↗
PubMed1994

Regulation of transducin GTPase activity in bovine rod outer segments.

The photoreceptor G-protein, transducin, belongs to the class of heterotrimeric GTP-binding proteins that transfer information from activated seven-span membrane receptors to effector enzymes or ion channels. Like other G-proteins, transducin acts as a molecular clock. It is activated by photoexcited rhodopsin which catalyzes the exchange of transducin-bound GDP for GTP and then stays active until bound GTP is hydrolyzed by an intrinsic GTPase activity. Our previous study on the components of the amphibian phototransduction cascade (Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417) has shown that transducin GTPase can be significantly accelerated by the target enzyme, cGMP phosphodiesterase (PDE), and more specifically its gamma-subunit (PDE gamma). Here we report that an analogous mechanism is present in bovine photoreceptors. Addition of recombinant PDE gamma to the test photoreceptor membranes which retain transducin but are depleted of endogenous PDE causes a significant acceleration of transducin GTPase activity. A similar effect was observed with the PDE holoenzyme, but not with the complex of PDE alpha- and beta-subunits prepared by a limited proteolysis of PDE with trypsin. The activating effect of PDE gamma is increased as test membrane concentration increases, exceeding 20-fold at rhodopsin concentrations over 80 microM and approaching the rate of the photoresponse turnoff. This suggests either that photoreceptor membranes contain a further factor which is essential for PDE-dependent regulation of transducin-bound GTP hydrolysis or that components of the phototransduction cascade interact in a cooperative manner. We also report that the GTPase-activating epitope is located within the C-terminal third of PDE gamma: the peptide corresponding to the 25 C-terminal amino acid residues of PDE gamma can accelerate transducin GTPase almost as well as the full-length PDE gamma. A part of the GTPase activating epitope is located within the 3 C-terminal amino acid residues: the truncation PDE gamma mutant lacking these residues accelerates transducin GTPase considerably less than the whole length PDE gamma.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1994

Structural determinants for activation of the alpha-subunit of a heterotrimeric G protein.

The 1.8 A crystal structure of transducin alpha.GDP, when compared to that of the activated complex with GTP-gamma S, reveals the nature of the conformational changes that occur on activation of a heterotrimeric G-protein alpha-subunit. Structural changes initiated by direct contacts with the terminal phosphate of GTP propagate to regions that have been implicated in effector activation. The changes are distinct from those observed in other members of the GTPase superfamily.

Amino Acid Sequence↗
PubMed1994

Arrestin-rhodopsin interaction. Multi-site binding delineated by peptide inhibition.

Visual arrestin modulates the intracellular response of retinal rod cells to light by specifically binding to the phosphorylated light-activated form of the photoreceptor rhodopsin (P-Rh*). In order to characterize the molecular interaction between rhodopsin and arrestin, we have studied the ability of synthetic peptides from the proposed cytoplasmic loops of rhodopsin to inhibit arrestin binding. A third cytoplasmic loop peptide competed most effectively for arrestin binding to P-Rh*, exhibiting an IC50 of 34 microM, while a first cytoplasmic loop peptide weakly inhibited binding with an IC50 of approximately 1100 microM. The first and third cytoplasmic loop peptides also inhibited P-Rh* interaction with both ARR[delta (2-16)-404], an arrestin mutant that lacks residues 2-16, and ARR[1-191], a mutant that contains only the amino half of arrestin. However, the third loop peptide had an approximately 5-fold lower affinity at inhibiting the binding of ARR[1-191] to P-Rh*. While the first and third loop peptides also inhibited arrestin binding to light-activated rhodopsin and a truncated rhodopsin lacking its C-terminal sites of phosphorylation, the peptides modestly enhanced arrestin binding to phosphorylated dark rhodopsin. These results suggest that the third and, to a lesser extent, the first cytoplasmic loops of rhodopsin may play an important role in arrestin binding to light-activated forms of rhodopsin.

Amino Acid Sequence↗
PubMed1993

The 2.2 A crystal structure of transducin-alpha complexed with GTP gamma S.

The 2.2 A crystal structure of activated rod transducin, Gt alpha.GTP gamma S, shows the bound GTP gamma S molecule occluded deep in a cleft between a domain structurally homologous to small GTPases and a helical domain unique to heterotrimeric G proteins. The structure, when combined with biochemical and genetic studies, suggests: how an activated receptor might open this cleft to allow nucleotide exchange; a mechanism for GTP-induced changes in effector and receptor binding surfaces; and a mechanism for GTPase activity not evident from previous data.

Amino Acid Sequence↗
PubMed1993

A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit.

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Sites of interaction between rod G-protein alpha-subunit and cGMP-phosphodiesterase gamma-subunit. Implications for the phosphodiesterase activation mechanism.

In photoreceptor cells of vertebrates light activates a series of protein-protein interactions resulting in activation of a cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form of rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is a key event for effector enzyme activation. This interaction has been studied using P gamma labeled with the fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 (P gamma LY) and sites of interaction on alpha t and P gamma have been investigated. Addition of alpha tGTP gamma S to P gamma LY produced a 3.2-fold increase in the fluorescence of P gamma LY. The Kd for alpha tGTP gamma S.P gamma LY interaction was 36 nM. Addition of 1 microM alpha tGDP had no effect, but in the presence of A1F4-, alpha tGDP increased P gamma LY fluorescence by 85%. When P gamma LY was reconstituted with P alpha beta to form fluorescent holo-PDE, alpha tGTP gamma S increased the fluorescence of holo-PDE with a K0.5 = 0.7 microM. Also, alpha tGTP gamma S stimulated the activity of this PDE over an identical range of concentrations with a similar K0.5 (0.6 microM). alpha tGTP gamma S enhanced the fluorescence of a COOH-terminal P gamma fragment, P gamma LY-46-87, as well (Kd = 1.5 microM). We demonstrate that an alpha t peptide, alpha t-293-314, which activated PDE (Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033), mediates PDE activation by interacting with the P gamma-46-87 region. Peptide alpha t-293-314 bound to P gamma LY (K0.5 = 1.2 microM) as well as to the carboxyl-terminal P gamma fragment, P gamma LY-46-87 (K0.5 = 1.7 microM) as measured by fluorescence increase, while other alpha t peptides had no effect. A peptide from the P gamma central region, P gamma-24-46, blocked the interaction between alpha tGTP gamma S and P gamma LY. The Kd for alpha tGTP gamma S.P gamma-24-46 interaction was 0.7 microM. On the other hand, P gamma-24-46 had no effect on alpha t-293-314 interaction with P gamma LY. Our data suggest that there are at least two distinct sites of interaction between alpha tGTP gamma S and P gamma. The interaction between alpha t-293-314 and P gamma-46-87 is important for PDE activation.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

A site on rod G protein alpha subunit that mediates effector activation.

The heterotrimeric guanine nucleotide binding proteins (G proteins) are activated by sensory or hormone receptors. In turn, the G proteins activate effector proteins such as adenylyl cyclase, cyclic guanosine 3',5'-monophosphate phosphodiesterase (cGMP PDE), phospholipase C, and potassium and calcium ion channels by mechanisms that are poorly understood. A site on the alpha subunit of the G protein transducin (alpha t) has been identified that interacts with and activates cGMP phosphodiesterase, the effector enzyme in rod photoreceptors. A 22-amino acid peptide, corresponding to residues 293 to 314 from the COOH-terminal region of alpha t, fully mimicked alpha t and potently activated PDE. This region is adjacent to the receptor activation domain; thus, the alpha subunit of this G protein has a site for interaction with both its effector and receptor that maps near the COOH-terminus.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Calmodulin binding distinguishes between beta gamma subunits of activated G proteins and transducin.

The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.

Adenylyl Cyclase Inhibitors↗
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↗
PubMed1991

A blue-light-activated GTP-binding protein in the plasma membranes of etiolated peas.

Heterotrimeric GTP-binding regulatory proteins (G proteins) have been identified as part of signal transduction systems in a wide variety of organisms. In this paper, we establish the presence of a G protein associated with the plasma membranes of the apical bud of etiolated peas. The GTPase activity is induced by low fluences of blue light administered to plasma membrane-enriched fractions. The activity is not responsive to red-light irradiation and is specific for GTP. The threshold for the excitation of the GTPase activity in vitro is less than 10(-1) mumol.m-2 of blue light, consistent with participation in the blue low-fluence system identified in the same tissue. A 40-kDa polypeptide is recognized by polyclonal antisera directed against the alpha subunit of the G protein transducin. The polypeptide also serves as a substrate for ADP-ribosylation by cholera and pertussis toxins. The ability of the 40-kDa polypeptide to serve as substrate for the toxin-mediated ribosylation is mediated by blue-light irradiation, implying that the 40-kDa polypeptide is the alpha subunit of a blue-light-stimulated G protein. The 40-kDa polypeptide binds a nonhydrolyzable photoaffinity-labeling analog of GTP only after irradiation with blue light. The protein we have described may function as an alpha subunit of a G protein active in the process of light-mediated development in higher plants.

Adenosine Diphosphate Ribose↗