Miles D. Houslay

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp19 and Trp20, whose orientation is optimized for bilayer insertion by the Leu16:Val17 pairing. Ca2+, at submicromolar levels, interacts with Asp21 in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp21−:Ca2+:PA2−:Lys24+) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidicacid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.

The phorbol ester, TPA inhibits glucagon‐stimulated adenylate cyclase activity

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour‐promoting phorbol ester TPA (12‐O‐tetradecanoyl phorbol‐13‐acetate). The half‐maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon‐stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 μg/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon‐stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.

Exchange of partners in glucagon receptor-adenylate cyclase complexes. Physical evidence for the independent, mobile receptor model

Abstract The apparent target sizes of the glucagon receptor and the catalytic unit of adenylate cyclase in rat liver plasma membranes have been measured by the technique of radiation inactivation in an electron beam. When irradiated in the uncoupled state, the apparent target size for the catalytic unit assayed by fluoride-stimulated activity was 160 000, and for the receptor assayed by specific 125 I-labelled glucagon binding was 217 000. The corresponding target size estimated from glucagon-stimulated activity after irradiation in the uncoupled state was 389 000. When the complexes were irradiated in the coupled state in the presence of glucagon, the apparent target sizes from 125 I-labelled glucagon binding, and fluoride- or glucagon-stimulated activities had similar values of 310 000, 380 000 and 421 000, respectively. However, if the complexes were allowed to uncouple by removing glucagon after irradiation and activity was then assayed after readdition of glucagon, the apparent target size from the glucagon-stimulated activity increases from 421 000 to 811 000. The pattern of apparent target sizes obtained under these different conditions has been tested against the pattern predicted for simple models of the coupling mechanism. The only simple model that is consistent with the pattern of target sizes requires the receptors and catalytic units to be present in approximately equal numbers. On binding glucagon, the receptor forms a locking interaction with the catalytic units, so that the complex and its components are inactivated as a single target with an apparent size of about 380 000 ( ± 15% ). After the removal and readdition of glucagon to complexes that were irradiated in the coupled state, the new population of complexes must contain hybrids of active and inactive partners obtained by exchange between active and inactivated complexes, to account for the doubling in apparent target size to 811 000 for glucagon-stimulated activity. This hybridization of catalytic units and receptors is the essential feature of the model that distinguishes it from others in which permanently associated complexes of the two components are activated by lateral dimersation on binding glucagon. Simple models of this type are shown to be physically improbable. It is emphasized that the models described are based only on the relationships between the apparent target sizes of components that are defined by their functions, and the apparent target sizes do not necessarily relate solely to the components that can be defined structurally as the receptor or catalytic unit.

The Activity of Adenylate Cyclase Is Regulated by the Nature of Its Lipid Environment

Publisher Summary The chapter describes the organizational aspects of the interactions among hormone receptors, the guanine nucleotide coupling protein, and adenylate cyclase as they pertain to the interpretation of lipid- mediated effects. Lipid fluidity has a marked effect on the steady state activity of adenylate cyclase, presumably by altering the physical constraints imposed upon the enzyme by the bilayer. However, it is also demonstrated that the fluidity of the bilayer can influence the rate of activation of turkey erythrocyte adenylate cyclase by β-agonists. This would be consistent with the need for the components to undergo independent lateral diffusion within the plane of the bilayer before functional collisions occurred. However, as the full activation of adenylate cyclase by hormones occurs within seconds, then the metabolically relevant effects of fluidity are going to be predominantly on the steady-state activity of the enzymes and not on the rates of activation of the enzyme. The role that bilayer fluidity plays in determining the activity of adenylate cyclase in its membrane environment is discussed.

Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity.

Both forskolin and ethanol elicit the activation of basal and ligand-stimulated adenylate cyclase activities in rat liver plasma membranes. Ethanol is most potent at activating the fluoride- and glucagon-stimulated activities whilst having little effect on basal activity. In contrast forskolin exerts its greatest effect on basal activity. Over the concentration range that ethanol activates adenylate cyclase, it also increases bilayer fluidity as indicated by a decrease in the values of the order parameters for an incorporated fatty acid spin probe. At high concentrations forskolin does increase bilayer fluidity. However, it only begins to do so at concentrations above those where forskolin has already exerted its maximal effect in activating adenylate cyclase. Forskolin can still activate, albeit to a reduced extent, detergent-solubilized adenylate cyclase whereas ethanol cannot. Forskolin elicits a pronounced rise in hepatocyte intracellular cyclic AMP concentrations, whereas ethanol does not. Both forskolin and ethanol reduce the temperature of onset of the lipid phase separation occurring in rat liver plasma membranes. This is detected in Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and order parameters of an incorporated fatty acid spin probe, where we find that forskolin is particularly potent in decreasing the temperature at which this lipid phase separation occurs. Our results are consistent with the notion that forskolin exerts its effect on adenylate cyclase primarily by a direct action on the catalytic unit of the enzyme. However, as forskolin is a potent perturber of the organisation of the lipid bilayer it is possible that this could modulate its effect on adenylate cyclase and might be expected to affect the activity of other membrane enzymes.

Insulin: in search of a mechanism

Several properties of Ln 3+ suggest a potential therapeutic role in conditions involving inflammation and excessive breakdown of connective tissue. These properties include the inhibition of col- lagenase, the stabilization of collagen fibrils, and the inhibition of lymphocyte activation, stimulus-mediated cell secre- tion and neutrophil chemotaxis and aggregation. Arthritis is well suited for further investigation of this potential, as Ln 3+ can be introduced by local intra- articular injection, thereby obviating the problems of toxicity when administered by the intravenous route, and poor oral absorption. Furthermore, Ln 3+ bind tightly to cartilage, thus retaining the agent in the desired location and minimizing pos- sible systemic complications. Preliminary results from experimental arthritis in rabbits are encouraging. References A brief reference list is a requirement for

A model for the selective mode of action of the irreversible monoamine oxidase inhibitors clorgyline and deprenyl, based on studies of their ability to activate a Ca2+‐Mg2+ ATPase in defined lipid environments

Clorgyline and deprenyl activated the pure Ca2+‐Mg2+ ATPase from rabbit muscle sarcoplasmic reticulum when it was in a defined lipid environment of either dimyristoyl lecithin or dipalmitoyl lecithin, apparently by fluidizing the phospholipid that surrounds the protein (the phospholipid annulus). Activation was only significant at temperatures when the annulus placed rigid constraints upon the protein. There was a two order of magnitude difference in the concentration at which the drugs achieved their effect which is thought to be related to their abilities to perturb the phospholipid annulus since it could not be attributed simply to the small differences in their partition coefficients. Clorgyline, which was more potent than deprenyl at effecting fluidization of the phospholipid annulus of the Ca2+‐Mg2+ ATPase in the defined lipid environments, activated species ‘B’ monoamine oxidase at concentrations at which it began to inhibit the species ‘A’ enzyme. Deprenyl did not activate the species ‘A’ enzyme at any concentration tested. It is suggested that, in tissues where there are multiple forms of mitochondrial monoamine oxidase, the selective action of clorgyline and deprenyl arises from a modification of the single inhibitor binding site on a protein species to yield species ‘A’ enzyme and species ‘B’ enzyme. One form of this modification to give the species ‘A’ monoamine oxidase would be a masking of the inhibitor binding site by lipid such that it could be readily penetrated by clorgyline but less readily so by deprenyl. The other form of the modification would yield the species ‘B’ enzyme, which like the ‘chaotrope‐treated’ enzyme and presumably ‘nascent’ monoamine oxidase, would expose the inhibitor binding site to the aqueous environment in which deprenyl would be the more efficacious inhibitor.

5-Hydroxytryptamine is a substrate for both species of monoamine oxidase in beef heart mitochondria

The activity of beef heart mitochondrial monoamine oxidase towards 5‐hydroxytryptamine (5‐HT) is inhibited by the selective inhibitors clorgyline, PCO [5‐phenyl‐3‐(N‐cyclopropyl)‐ethylamine‐1,2,4‐oxadiazole] and Deprenyl with a biphasic dependence on the inhibitor concentration. The activities towards tyramine, dopamine and tryptamine were also inhibited in a biphasic manner, but the apparent proportions of the two enzyme species active on dopamine and tryptamine depended on the inhibitor used. Phenethylamine oxidation was inhibited in a monophasic manner suggesting that only a single enzyme species was responsible for the oxidation of this substrate. The biphasic response of 5‐HT oxidation to inhibition by clorgyline persisted when functionally competent mitochondria were used and was unaffected by the soluble amine oxidase inhibitors semicarbazide and amino‐guanidine. These results indicate that the behaviour of the beef heart enzyme towards selective inhibitors is considerably different from that of any preparations previously studied and suggest that the classification of monoamine oxidase activities into A and B types may be only of limited usefulness.

Thermotropic lipid phase separations in human platelet and rat liver plasma membranes

SummaryElectron spin resonance (ESR) studies were conducted on human platelet plasma membranes using 5-nitroxide stearate, I(12,3). The polarity-corrected order parameterS and polarity-uncorrected order parametersS(T‖) andS(T⊥) were independent of probe concentration at low I(12,3)/membrane protein ratios. At higher ratios,S andS(T⊥) decreased with increasing probe concentration whileS(T‖) remained unchanged. This is the result of enhanced radical interactions due to probe clustering. A lipid phase separation occurs in platelet membranes that segregates I(12,3) for temperatures less than 37°C. As Arrhenius plots of platelet acid phosphatase activity exhibit a break at 35 to 36°C, this enzyme activity may be influenced by the above phase separation. Similar experiments were performed on native [cholesterol/phospholipid ratio (C/P)=0.71] and cholesterol-enriched [C/P=0.85] rat liver plasma membranes. At 36°C, cholesterol loading reduces I(12,3) flexibility and decreases the probe ratio at which radical interactions are apparent. The latter effects are attributed to the formation of cholesterol-rich lipid domains, and to the inability of I(12,3) to partition into these domains because of steric hinderance. Cholesterol enrichment increases both the high temperature onset of the phase separation occurring in liver membranes from 28° to 37°C and the percentage of probe-excluding, cholesterolrich lipid domains at elevated temperatures. A model is discussed attributing the lipid phase separation in native liver plasma membranes to cholesterol-rich and-poor domains. As I(12,3) behaves similarly in cholesterol-enriched liver and human platelet plasma membranes, cholesterol-rich and-poor domains probably exist in both systems at physiologic temperatures.

Guanine nucleotides can activate the insulin‐stimulated phosphodiesterase in liver plasma membranes

The insulin‐stimulated cyclic AMP phosphodiesterase from liver plasma membranes is shown to be activated upon incubation with guanine nucleotides in the presence of ATP. The non‐hydrolysable analogue of ATP, adenylyl imidodiphosphate failed to substitute for ATP in achieving activation. GTP, its non‐hydrolysable analogues p[NH]ppG and GTP‐γ‐S, as well as GDP, all elicited activation. It is suggested that guanine nucleotides, and probably insulin, exert their effect on this enzyme through a distinct species of guanine nucleotide regulatory protein.