Biomedical subjects
Publications and source records attributed to L E Limbird.
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The molecular size of adenylate cyclase solubilized from frog erythrocyte membranes by digitonin extraction has been determined by chromatography on Sepharose 6B. Regardless of whether the membranes are exposed to catecholamines, GPP(NH)P, NaF or no effector prior to solubilization, the apparent molecular size of the adenylate cyclase enzyme is the same. Furthermore, a similar elution profile for the enzyme is observed when the catalytic activity in the eluates is measured in the presence of Mn++, rather than Mg++. Since it is generally assumed that the persistent activation of adenylate cyclase by GPP(NH)P requires interaction of the catalytic moiety with the guanine nucleotide regulatory site, it appears that the adenylate cyclase activity detected in the column eluates represents an intact catalytic-regulatory site complex. The adenylate cyclase activity derived from catecholamine pretreated frog erythrocyte membranes does not co-elute with catecholamine-occupied beta-adrenergic receptors, indicating that the agonist-promoted increase in apparent receptor size observed here and in earlier studies does not represent a physical coupling of the receptor and the adenylate cyclase enzyme.
Direct radioligand binding studies have been used to probe the molecular mechanisms whereby agonist catecholamines regulate the function of beta-adrenergic receptors in a model system, the frog erythrocyte. The unique characteristics of agonist as opposed to antagonist action are first, the ability to stimulate the adenylate cyclase through the receptor and second, the ability to desensitize the system by alterations induced in beta-adrenergic receptors. These properties of agonist are not shared by antagonist despite the high affinity and specificity of antagonist binding to the beta-adrenergic receptors. Agonist and antagonist receptor complexes may be distinguished in a variety of ways including differences in their sensitivity to regulatory guanine nucleotides and also by gel chromatography on AcA 34 Ultragel. The agonist receptor complex appears to elute from the columns with an apparently increased size. A "dynamic receptor affinity model" of beta-adrenergic receptor action is proposed which features several distinct conformational states of the receptor. Agonists have much higher affinity for the physiologically active or coupled state of the receptor, whereas antagonists have equal affinity for both. In addition, a third "desensitized" state of the receptor is also postulated to exist.
The properties of digitonin-solubilized beta-adrenergic receptors from frog erythrocyte membranes were studied by gel exclusion chromatography on AcA 34 Ultragel. beta-Adrenergic receptor binding activity in these membranes can be identified by both an agonist ligand, [(3)H]hydroxybenzylisoproterenol, and the antagonist ligands, [(3)H]dihydroalprenolol and (125)I-labeled hydroxybenzylpindolol. Occupancy of the beta-adrenergic receptors with the [(3)H]hydroxybenzylisoproterenol agonist prior to their solubilization from the membrane leads to an increase in apparent receptor size. Alterations in the molecular size of the receptor cannot be mimicked by occupancy of the binding site with the antagonist ligands. Exposure of frog erythrocyte membranes to [(3)H]hydroxybenzylisoproterenol agonist in the presence of 10 muM Gpp(NH)(p), a guanyl nucleotide analog that exerts multiple regulatory effects on the catecholamine-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] system, results in the elution of the [(3)H]hydroxybenzylisoproterenol radioligand in both the region characteristic of the agonist-receptor complex and the region characteristic of the antagonist-receptor complex. The precise molecular interactions responsible for the agonist-induced increase in apparent beta-adrenergic receptor size are still unresolved. However, the low concentrations of agonist that are capable of altering apparent receptor size and the sensitivity of this effect to guanyl nucleotides suggest that these phenomena may be intimately involved in eliciting the physiological effects of beta-adrenergic catecholamines at the molecular level.
The frog erythrocyte membrane provides an excellent model system for the study of beta-adrenergic receptor-adenylate cyclase interactions since it possesses an adenylate cyclase enzyme which is very responsive to catecholamines. The purpose of these studies was to evaluate directly whether the functions of receptor binding and adenylate cyclase activity are carried out by a single macromolecule or separable molecular entities. Obtaining this information is a first step in understanding at a molecular level how receptor binding is "coupled" to enzyme activation. Binding and cyclase activities were solubilized from the frog erythrocyte membrane with digitonin and were observed to partition independently during gel exclusion chromatography in the presence of solubilizing detergent. This finding documents that the beta-adrenergic receptor and adenylate cyclase enzyme are, in fact, separable macromolecules. Under the particular experimental conditions employed, the elution of beta-adrenergic receptor binding on Sepharose 6B was not altered by the absence or presence of beta-adrenergic agonist or antagonist ligands or by exposure of the membranes prior to solubulization to the guanyl nucleotide analog, guanyl-5'-yl imidodiphosphate.
(-)-[3H]Dihydroalprenolol, a potent competitive beta-adrenergic antagonist, has been previously documented to bind to the adenylate cyclase-coupled beta-adrenergic receptor sites in mammalian and non-mammalian tissues. Steady state binding of (-)-[3H]dihydroalprenolol to sites in frog erythrocyte membranes, a model system for adenylate cyclase-coupled beta-adrenergic receptors, displays characteristics consistent with negative cooperativity among the beta-adrenergic receptors: Scatchard plots are curvilinear with upward concavity and slopes of Hill plots are consistently less than 1.0. The existence of site-site interactions of the negatively cooperative type were demonstrated directly by the ability of unlabeled (-)-alprenolol to accelerate the dissociation of (-)-[3H]dihydroalprenolol under conditions were no rebinding of radioligand occurred. The dissociation rate of (-)-[3H]dihydroalprenolol alone is directly related to temperature and increases with increases in temperature from 4-37 degrees. (-)-[3H]Dihydroalprenolol dissociation is enhanced by unlabeled (-)-alprenolol at all temperatures studied; however, at 4 degrees, the time required to observe an enhancement of radioligand dissociated is greater than the time required for unlabeled (-)-alprenolol to occupy the empty receptor sites, suggesting that increased rigidity of the biomembrane at 4 degrees may be responsible for the absence of readily observable site-site interactions. The ability of a number of beta-adrenergic agonists and antagonists to induce negative cooperativity among the beta-adrenergic receptors was directly related to their affinity for the receptor sites rather than their intrinsic activity in the adenylate cyclase-coupled beta-adrenergic system. The ability to induce site-site interactions among the beta-adrenergic receptors occurs at physiological concentrations of beta-adrenergic agents, since occupancy of less than 10% of the receptor sites is sufficient to reduce receptor affinity. Changes in pH from 6.5 to 9.0 did not significantly alter the negatively cooperative site-site interactions among the receptor sites. The negatively cooperative phenomenon was also independent of Mg2+, Ca2+, and NaF concentrations in the buffer medium. The presence of guanyl-5'-yl imidodiphosphate, a nonhydrolyzable nucleotide analog which enhances adenylate cyclase stimulation (Vmax) by beta-adrenergic agonists and decreases the concentration of agonist required to half-maximally stimulate adenylate cyclase, did not alter the ability of either agonists or antagonists to induce negatively cooperative site-site interactions among the beta-adrenergic receptors.
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The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.
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Localization of receptors in discrete cellular microdomains undoubtedly contributes to their interaction with particular effectors and receptor targets. For G protein-coupled receptors, virtually nothing is known about the mechanisms and structural features responsible for their targeting to and retention in varying surface domains. We have shown that the Gi/ Go-coupled alpha 2A-adrenergic receptor (alpha 2AAR) is directly targeted to the lateral subdomain of MDCK II cells. Mutational analysis has revealed that regions in or near the bilayer are likely critical for alpha 2AAR targeting, whereas endofacial domains contribute to alpha 2AAR retention on the lateral surface. Although the alpha 2BAR also is enriched on the lateral subdomain at steady-state, its polarization occurs after initial random delivery to both apical and basolateral surfaces followed by a selective accumulation on the lateral subdomain. The alpha 2CAR also is expressed on the lateral subdomain and achieves its localization via direct delivery to the basolateral surface; however, the alpha 2CAR also exists in an as yet not fully characterized intracellular compartment. Interestingly, another Gi/Go-coupled receptor, the A1 adenosine receptor, is enriched on the apical surface of MDCK II calls and achieves this localization by direct apical delivery. These findings indicate that receptor delivery to polarized surfaces is not determined by receptor coupling to a specific subpopulation of G proteins.