Since recent reports suggest that endocytosis is required for some G proteincoupled receptors to ... more Since recent reports suggest that endocytosis is required for some G proteincoupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M.,
The selective a 2 -adrenergic receptor antagonist rauwolscine was structurally modified to yield ... more The selective a 2 -adrenergic receptor antagonist rauwolscine was structurally modified to yield a series of arylamine carboxamide derivatives, which were investigated as potential molecular probes for the localization and structural characterization of a 2 -adrenergic receptors. The arylamine carboxamides differ in the number of carbon atoms separating the reactive phenyl moiety from the fused ring structure of the parent compound, rauwolscine carboxylate. Competitive inhibition studies with [ 3 H]rauwolscine in rat kidney membranes indicate that the affinity for the carboxamide derivatives is inversely related to the length of the carbon spacer arm with rauwolscine 4-aminophenyl carboxamide (zero carbon spacer arm; rau-AMPC) exhibiting the highest affinity (K A = 2.3 ± 0.2 nM). Radioiodination of rau-AMPC yields a ligand, 125 I-rau-AMPC, which binds to rat kidney a 2adrenergic receptors with high affinity, as determined by both kinetic analysis (K t = k 2 /k, = 0.016 min-'/2.1 x HTM-1 min-' = 0.76 nM) and equilibrium binding studies (K ? = 0.78 ± 0.16 nM). 12S Irau-AMPC was quantitatively converted to the photolabile arylazide derivative 17a-hydroxy-20ayohimban-16/3-(JV-4-azido-3-[ I]iodophenyl)carboxamide ( 12S I-rau-AZPC). In a partially purified receptor preparation from porcine brain, this compound photolabels a major (M r = 62,000) peptide. The labeling of this peptide is inhibited by adrenergic agonists and antagonists with a rank order of potency consistent with an aj-adrenergic receptor binding site. Both l25 I-rau-AMPC and the photolabile arylazide derivative, l2S I-rau-AZPC, should prove useful as molecular probes for the structural and biochemical characterization of a 2 -adrenergic receptors. (Hypertension 9 [Suppl III]: III-120-IIM24, 1987) KEY WORDS brain a 2 -adrenergic receptors * photoaffinity label • rauwolscine • porcine A LPHA 2 -adrenergic receptors are located at pre-/ \ junctional and postjunctional sites in the cen-A. \ ~ tral nervous system and in several peripheral organs, including heart, kidney, and pancreatic islets. 1 These cell membrane glycoproteins mediate a number of diverse effects in the body, such as vasoconstric-tion, inhibition of insulin release, platelet aggregation, and various events triggered in the central nervous system. 1 Several studies suggest that a 2 -adrenergic receptors differ in various tissues and species with regard to signal transduction mechanisms and ligand recognition
Previous studies in cultured, polarized Madin-Darby canine kidney II (MDCKII) renal epithelial ce... more Previous studies in cultured, polarized Madin-Darby canine kidney II (MDCKII) renal epithelial cells have demonstrated that the apical steady-state localization and delivery of the A(1) adenosine receptor is modified by disruption of the microtubule network with colchicine, whereas the basolateral localization and trafficking of the alpha(2)-adrenergic receptors (alpha(2)AR) are not; instead, the binding capacity of the alpha(2B)AR, but not alpha(2A)AR or alpha(2C)AR subtypes, is increased in a time-dependent fashion. The present studies explore the molecular basis for this alpha(2B)AR subtype-selective phenomenon. Colchicine selectively increased alpha(2B)AR density at the cell surface, as determined by confocal microscopy, receptor binding, and surface biotinylation studies. The colchicine-induced increase in the functional density of the alpha(2B)AR requires the third intracellular loop because the alpha(2B)AR loop deletion (alpha(2B)ARtriangle upi3) mutant did not show an increased receptor density after colchicine treatment. Furthermore, the colchicine-mediated increase in alpha(2B)AR density is manifest only in polarized cells because colchicine treatment of nonpolarized MDCKII renal epithelial cells as well as simian kidney COSM6 and human embryonic kidney HEK293 cells did not effect an increase in alpha(2B)AR density. Colchicine-dependent increases in alpha(2B)AR density did not depend on functional coupling to G proteins, however, because pretreatment with pertussis toxin did not eliminate the effect of colchicine. These data indicate that microtubule-dependent regulation of alpha(2B)AR density at the basolateral surface of polarized MDCKII cells requires the third intracellular loop of alpha(2B)AR but not functional alpha(2B)AR-G protein coupling.
The syndrome of mid-systolic click (MSC) and late systolic murmur (LSM) is associated with mild m... more The syndrome of mid-systolic click (MSC) and late systolic murmur (LSM) is associated with mild mitral regurgitation. Since patients with MSC and LSM often have atypical chest pain, abnormal ECGs and arrhythmias, we evaluated the effect of increasing systol ic arterial pressure on these signs and symptoms. The phonocardiogram, KG and BP were monitored before and during introvenous phenylephrine (P) infusion (33pg/min) in 5 women (age 19
Extensive trypsinization of the purified alpha 2-adrenergic receptor and repurification by wheat ... more Extensive trypsinization of the purified alpha 2-adrenergic receptor and repurification by wheat germ agglutinin-agarose chromatography yields an adrenergic ligand-binding hydrophobic core of the receptor. Allosteric modulation of adrenergic ligand binding by Na+, H+, and 5-amino-substituted analogs of amiloride is quantitatively retained in this core, as assessed by the ability of these agents to accelerate the rate of [3H] yohimbine dissociation from the adrenergic ligand-binding site. These findings refine our understanding of where within the alpha 2-adrenergic receptor structure these allosteric agents bind and, for the effects of Na+ and H+, allow certain predictions to be made as to which carboxylic acid side chains are probable candidates for participation in a monovalent cation-binding pocket within the hydrophobic tryptic core of the receptor.
The noradrenergic system is involved in the regulation of many physiological and psychological pr... more The noradrenergic system is involved in the regulation of many physiological and psychological processes, including the modulation of mood. The alpha(2)-adrenergic receptors (alpha(2)-ARs) modulate norepinephrine release, as well as the release of serotonin and other neurotransmitters, and are therefore potential targets for antidepressant and anxiolytic drug development. The current studies were undertaken to examine the role of the alpha(2A) subtype of alpha(2)-AR in mouse behavioral models of depression and anxiety. We have observed that the genetic knock-out of the alpha(2A)-AR makes mice less active in a modified version of Porsolt's forced swim test and insensitive to the antidepressant effects of the tricyclic drug imipramine in this paradigm. Furthermore, alpha(2A)-AR knock-out mice appear more anxious than wild-type C57 Bl/6 mice in the rearing and light-dark models of anxiety after injection stress. These findings suggest that the alpha(2A)-AR may play a protective role in some forms of depression and anxiety and that the antidepressant effects of imipramine may be mediated by the alpha(2A)-AR.
The maturation of rat reticulocytes to erythrocytes is paralleled by a marked loss of adenylate c... more The maturation of rat reticulocytes to erythrocytes is paralleled by a marked loss of adenylate cyclase responsiveness to catecholamines and guanine nucleotides without significant loss of basal enzyme activity. These changes in adenylate cyclase activity are accompanied by a parallel %fold decrease in both the number of p-adrenergic binding sites and the quantity of M, = 42,000 protein substrates for cholera toxin-catalyzed [32P]ADP ribosylation. In addition, the maturation of the rat erythrocyte is accompanied by a loss of the ability of guanine nucleotides to modulate receptoragonist interactions. Thus, whereas guanine nucleotides promote tritiated hydroxybenzylisoproterenol agonist dissociation from the P-adrenergic receptor and reduce the potency of isoproterenol in competing for dihydroalprenolol antagonist binding in rat reticulocyte membranes, these regulatory effects of guanine nucleotides are virtually absent in membranes derived from mature rat erythrocytes. Furthermore, the ability of agonist occupancy of the P-adrenergic receptor of rat reticulocyte membranes to promote an increase in receptor size, as assessed by gel exclusion chromatography of solubilized preparations, is not observed in mature erythrocyte preparations. This agonist-induced increase in apparent receptor size has been previously observed in frog erythrocyte membranes (Limbird, L. E., and Lefkowitz, R. J. (1978) Proc. NatZ. Acad. Sei. U. S. A. 75,228-232) and is thought to reflect the formation of a receptor guanine nucleotide regulatory complex. Consistent with this hypothesis is the ability of guanine nucleotides to reverse the formation of this larger molecular size form of the receptor in rat reticulocyte membranes. The inability to promote this receptorguanine nucleotide regulatory protein complex in rat erythrocyte membranes suggests that one molecular concomitant of rat erythrocyte maturation is a decrease in the effectiveness of receptor-guanine nucleotide regulatory protein communication.
We have mutated the aspartate residue in the putative second transmembrane spanning domain of the... more We have mutated the aspartate residue in the putative second transmembrane spanning domain of the alpha 2A-adrenergic receptor (alpha 2AAR) to the non-negatively charged asparagine (D79N) and glutamine (D79Q) and the negatively charged glutamate (D79E) residue in an effort to better characterize the role of this residue, highly conserved among G-protein-coupled receptors, in Na+ regulation of ligand binding and in receptor G-protein coupling. Allosteric modulation of receptor-ligand interactions by Na+ is retained by the D79E alpha 2AAR but lost upon mutation to the uncharged D79N and D79Q residues. Loss of allosteric effects of Na+ is paralleled by a complete loss of retrograde information transfer from G-proteins to alpha 2AAR in AtT20 cells, measured via the sensitivity of radiolabeled agonist binding to Gpp(NH)p. In contrast to the complete elimination of retrograde signaling via the D79N and D79Q alpha 2AAR, anterograde information transfer from receptor to G-protein is modified in a more subtle quantitative way, since agonist-stimulated GTPase activity via D79N and D79Q alpha 2AAR, although apparently attenuated compared to wild type and D79E alpha 2AAR, is no less than the GTPase activity elicited by endogenous somatostatin receptors in AtT20 cells. These data indicate that a negative charge at amino acid residue 79 forecasts sensitivity to allosteric regulation by monovalent cations and its mutation to non-negatively charged residues elicits a nonparallel modulation of receptor-->G-protein versus G-protein-->receptor communication between alpha 2AAR and pertussis toxin-sensitive GTP-binding proteins.
A high capacity, ..cap alpha../sub 2/AR-selective affinity resin (YOH. ag) has been prepared by c... more A high capacity, ..cap alpha../sub 2/AR-selective affinity resin (YOH. ag) has been prepared by coupling yohimbinic acid to diaminodipropylamine agarose with 1,3 dicyclohexylcarbodiimide. Unreacted amino groups on the agarose matrix are blocked subsequently by acetylation. One volume of YOH. ag adsorbs 75% of the ..cap alpha../sub 2/AR from 50 volumes of digitonin-solubilized preparation containing 0.2 pmol ..cap alpha../sub 2/AR/mg protein. Digitonin-solubilized preparations are derived from cholate extracts of porcine cerebral cortex containing approx. 0.075 pmol ..cap alpha../sub 2/AR/mg protein. Adsorption of ..cap alpha../sub 2/AR to YOH. ag is selective and thus is blocked by the ..cap alpha..-adrenergic antagonist phentolamine. Adsorbed ..cap alpha../sub 2/AR are eluted with 10 ..mu..M phentolamine (20% yield) after removal of non-related proteins with NaCl gradients. Following hydroxylapatite chromatography to concentrate ..cap alpha..''AR and to remove phentolamine, the ..cap alpha..AR is present at 200-400 pmol/mg protein, assayed using sub-saturating concentrations of (/sup 3/H)-yohimbine. (It is estimated that the specific activity of a homogeneous ..cap alpha../sub 2/AR preparation would be 12,000-16,000 pmol/mg protein.) The availability of large quantities of cortical ..cap alpha../sub 2/AR and a resin easily prepared from commercially-supplied reagents suggests that purification of quantities of ..cap alpha../sub 2/AR sufficient for subsequent biochemical studies is feasible.
Abstract. It has been suggested that-adren-ergic responsiveness is reduced in hypertension. To ev... more Abstract. It has been suggested that-adren-ergic responsiveness is reduced in hypertension. To eval-uate a possible alteration in human 3-receptors that might account for diminished /3-adrenergic responsiveness, we studied leukocytes from hypertensive and normotensive subjects after an overnight rest supine, and then after being ambulatory, a maneuver that increases plasma cat-echolamines approximately twofold. In supine samples, /-receptor affinity for the agonist isoproterenol was sig-nificantly reduced in hypertensives and was associated with a reduction in the proportion of/-receptors binding agonist with a high affinity from 42±6 % in normotensive subjects to 25±2 % in hypertensives (P < 0.05). Alterations in /3-adrenergic-mediated adenylate cyclase activity par-allelled the differences seen in the /-receptor affinity for agonist. In normotensive subjects, /-receptor density and the proportion of receptors binding agonist with high affinity were reciprocally correlated with ...
... Human Platelet AIpha2-Adrenergic Receptor in Membrane and Solubilized Preparations LEE E. LIM... more ... Human Platelet AIpha2-Adrenergic Receptor in Membrane and Solubilized Preparations LEE E. LIMBIRD,' JANICE L. SPECK, AND SHARON K. SMITH ... Despite the qualitatively different effects of Na on alpha2-receptor-agonist and alpha-2-receptor-antagonist inter-...
systems suggests that a common pool of GTP-binding proteins could be shared by hormonal systems c... more systems suggests that a common pool of GTP-binding proteins could be shared by hormonal systems coupled to activation and inhibition of the enzyme. Differential sensitivity to proteases (2, 3), Mn2+ (4, 5),
A Short Course on Theory and Methods, Second Edition is a primer for the study of cell surface re... more A Short Course on Theory and Methods, Second Edition is a primer for the study of cell surface receptors. The simplified discussion of methods and their underlying principles removes the usual intimidation caused by the specialized vocabulary or sophisticated mathematics that characterize many of the primary papers in this field. In this way, the basic concepts become emphasized. This volume is a starting point: a textbook as well as a manual to which the investigator can return for a refresher course, when needed.
Since recent reports suggest that endocytosis is required for some G proteincoupled receptors to ... more Since recent reports suggest that endocytosis is required for some G proteincoupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M.,
The selective a 2 -adrenergic receptor antagonist rauwolscine was structurally modified to yield ... more The selective a 2 -adrenergic receptor antagonist rauwolscine was structurally modified to yield a series of arylamine carboxamide derivatives, which were investigated as potential molecular probes for the localization and structural characterization of a 2 -adrenergic receptors. The arylamine carboxamides differ in the number of carbon atoms separating the reactive phenyl moiety from the fused ring structure of the parent compound, rauwolscine carboxylate. Competitive inhibition studies with [ 3 H]rauwolscine in rat kidney membranes indicate that the affinity for the carboxamide derivatives is inversely related to the length of the carbon spacer arm with rauwolscine 4-aminophenyl carboxamide (zero carbon spacer arm; rau-AMPC) exhibiting the highest affinity (K A = 2.3 ± 0.2 nM). Radioiodination of rau-AMPC yields a ligand, 125 I-rau-AMPC, which binds to rat kidney a 2adrenergic receptors with high affinity, as determined by both kinetic analysis (K t = k 2 /k, = 0.016 min-'/2.1 x HTM-1 min-' = 0.76 nM) and equilibrium binding studies (K ? = 0.78 ± 0.16 nM). 12S Irau-AMPC was quantitatively converted to the photolabile arylazide derivative 17a-hydroxy-20ayohimban-16/3-(JV-4-azido-3-[ I]iodophenyl)carboxamide ( 12S I-rau-AZPC). In a partially purified receptor preparation from porcine brain, this compound photolabels a major (M r = 62,000) peptide. The labeling of this peptide is inhibited by adrenergic agonists and antagonists with a rank order of potency consistent with an aj-adrenergic receptor binding site. Both l25 I-rau-AMPC and the photolabile arylazide derivative, l2S I-rau-AZPC, should prove useful as molecular probes for the structural and biochemical characterization of a 2 -adrenergic receptors. (Hypertension 9 [Suppl III]: III-120-IIM24, 1987) KEY WORDS brain a 2 -adrenergic receptors * photoaffinity label • rauwolscine • porcine A LPHA 2 -adrenergic receptors are located at pre-/ \ junctional and postjunctional sites in the cen-A. \ ~ tral nervous system and in several peripheral organs, including heart, kidney, and pancreatic islets. 1 These cell membrane glycoproteins mediate a number of diverse effects in the body, such as vasoconstric-tion, inhibition of insulin release, platelet aggregation, and various events triggered in the central nervous system. 1 Several studies suggest that a 2 -adrenergic receptors differ in various tissues and species with regard to signal transduction mechanisms and ligand recognition
Previous studies in cultured, polarized Madin-Darby canine kidney II (MDCKII) renal epithelial ce... more Previous studies in cultured, polarized Madin-Darby canine kidney II (MDCKII) renal epithelial cells have demonstrated that the apical steady-state localization and delivery of the A(1) adenosine receptor is modified by disruption of the microtubule network with colchicine, whereas the basolateral localization and trafficking of the alpha(2)-adrenergic receptors (alpha(2)AR) are not; instead, the binding capacity of the alpha(2B)AR, but not alpha(2A)AR or alpha(2C)AR subtypes, is increased in a time-dependent fashion. The present studies explore the molecular basis for this alpha(2B)AR subtype-selective phenomenon. Colchicine selectively increased alpha(2B)AR density at the cell surface, as determined by confocal microscopy, receptor binding, and surface biotinylation studies. The colchicine-induced increase in the functional density of the alpha(2B)AR requires the third intracellular loop because the alpha(2B)AR loop deletion (alpha(2B)ARtriangle upi3) mutant did not show an increased receptor density after colchicine treatment. Furthermore, the colchicine-mediated increase in alpha(2B)AR density is manifest only in polarized cells because colchicine treatment of nonpolarized MDCKII renal epithelial cells as well as simian kidney COSM6 and human embryonic kidney HEK293 cells did not effect an increase in alpha(2B)AR density. Colchicine-dependent increases in alpha(2B)AR density did not depend on functional coupling to G proteins, however, because pretreatment with pertussis toxin did not eliminate the effect of colchicine. These data indicate that microtubule-dependent regulation of alpha(2B)AR density at the basolateral surface of polarized MDCKII cells requires the third intracellular loop of alpha(2B)AR but not functional alpha(2B)AR-G protein coupling.
The syndrome of mid-systolic click (MSC) and late systolic murmur (LSM) is associated with mild m... more The syndrome of mid-systolic click (MSC) and late systolic murmur (LSM) is associated with mild mitral regurgitation. Since patients with MSC and LSM often have atypical chest pain, abnormal ECGs and arrhythmias, we evaluated the effect of increasing systol ic arterial pressure on these signs and symptoms. The phonocardiogram, KG and BP were monitored before and during introvenous phenylephrine (P) infusion (33pg/min) in 5 women (age 19
Extensive trypsinization of the purified alpha 2-adrenergic receptor and repurification by wheat ... more Extensive trypsinization of the purified alpha 2-adrenergic receptor and repurification by wheat germ agglutinin-agarose chromatography yields an adrenergic ligand-binding hydrophobic core of the receptor. Allosteric modulation of adrenergic ligand binding by Na+, H+, and 5-amino-substituted analogs of amiloride is quantitatively retained in this core, as assessed by the ability of these agents to accelerate the rate of [3H] yohimbine dissociation from the adrenergic ligand-binding site. These findings refine our understanding of where within the alpha 2-adrenergic receptor structure these allosteric agents bind and, for the effects of Na+ and H+, allow certain predictions to be made as to which carboxylic acid side chains are probable candidates for participation in a monovalent cation-binding pocket within the hydrophobic tryptic core of the receptor.
The noradrenergic system is involved in the regulation of many physiological and psychological pr... more The noradrenergic system is involved in the regulation of many physiological and psychological processes, including the modulation of mood. The alpha(2)-adrenergic receptors (alpha(2)-ARs) modulate norepinephrine release, as well as the release of serotonin and other neurotransmitters, and are therefore potential targets for antidepressant and anxiolytic drug development. The current studies were undertaken to examine the role of the alpha(2A) subtype of alpha(2)-AR in mouse behavioral models of depression and anxiety. We have observed that the genetic knock-out of the alpha(2A)-AR makes mice less active in a modified version of Porsolt's forced swim test and insensitive to the antidepressant effects of the tricyclic drug imipramine in this paradigm. Furthermore, alpha(2A)-AR knock-out mice appear more anxious than wild-type C57 Bl/6 mice in the rearing and light-dark models of anxiety after injection stress. These findings suggest that the alpha(2A)-AR may play a protective role in some forms of depression and anxiety and that the antidepressant effects of imipramine may be mediated by the alpha(2A)-AR.
The maturation of rat reticulocytes to erythrocytes is paralleled by a marked loss of adenylate c... more The maturation of rat reticulocytes to erythrocytes is paralleled by a marked loss of adenylate cyclase responsiveness to catecholamines and guanine nucleotides without significant loss of basal enzyme activity. These changes in adenylate cyclase activity are accompanied by a parallel %fold decrease in both the number of p-adrenergic binding sites and the quantity of M, = 42,000 protein substrates for cholera toxin-catalyzed [32P]ADP ribosylation. In addition, the maturation of the rat erythrocyte is accompanied by a loss of the ability of guanine nucleotides to modulate receptoragonist interactions. Thus, whereas guanine nucleotides promote tritiated hydroxybenzylisoproterenol agonist dissociation from the P-adrenergic receptor and reduce the potency of isoproterenol in competing for dihydroalprenolol antagonist binding in rat reticulocyte membranes, these regulatory effects of guanine nucleotides are virtually absent in membranes derived from mature rat erythrocytes. Furthermore, the ability of agonist occupancy of the P-adrenergic receptor of rat reticulocyte membranes to promote an increase in receptor size, as assessed by gel exclusion chromatography of solubilized preparations, is not observed in mature erythrocyte preparations. This agonist-induced increase in apparent receptor size has been previously observed in frog erythrocyte membranes (Limbird, L. E., and Lefkowitz, R. J. (1978) Proc. NatZ. Acad. Sei. U. S. A. 75,228-232) and is thought to reflect the formation of a receptor guanine nucleotide regulatory complex. Consistent with this hypothesis is the ability of guanine nucleotides to reverse the formation of this larger molecular size form of the receptor in rat reticulocyte membranes. The inability to promote this receptorguanine nucleotide regulatory protein complex in rat erythrocyte membranes suggests that one molecular concomitant of rat erythrocyte maturation is a decrease in the effectiveness of receptor-guanine nucleotide regulatory protein communication.
We have mutated the aspartate residue in the putative second transmembrane spanning domain of the... more We have mutated the aspartate residue in the putative second transmembrane spanning domain of the alpha 2A-adrenergic receptor (alpha 2AAR) to the non-negatively charged asparagine (D79N) and glutamine (D79Q) and the negatively charged glutamate (D79E) residue in an effort to better characterize the role of this residue, highly conserved among G-protein-coupled receptors, in Na+ regulation of ligand binding and in receptor G-protein coupling. Allosteric modulation of receptor-ligand interactions by Na+ is retained by the D79E alpha 2AAR but lost upon mutation to the uncharged D79N and D79Q residues. Loss of allosteric effects of Na+ is paralleled by a complete loss of retrograde information transfer from G-proteins to alpha 2AAR in AtT20 cells, measured via the sensitivity of radiolabeled agonist binding to Gpp(NH)p. In contrast to the complete elimination of retrograde signaling via the D79N and D79Q alpha 2AAR, anterograde information transfer from receptor to G-protein is modified in a more subtle quantitative way, since agonist-stimulated GTPase activity via D79N and D79Q alpha 2AAR, although apparently attenuated compared to wild type and D79E alpha 2AAR, is no less than the GTPase activity elicited by endogenous somatostatin receptors in AtT20 cells. These data indicate that a negative charge at amino acid residue 79 forecasts sensitivity to allosteric regulation by monovalent cations and its mutation to non-negatively charged residues elicits a nonparallel modulation of receptor--&gt;G-protein versus G-protein--&gt;receptor communication between alpha 2AAR and pertussis toxin-sensitive GTP-binding proteins.
A high capacity, ..cap alpha../sub 2/AR-selective affinity resin (YOH. ag) has been prepared by c... more A high capacity, ..cap alpha../sub 2/AR-selective affinity resin (YOH. ag) has been prepared by coupling yohimbinic acid to diaminodipropylamine agarose with 1,3 dicyclohexylcarbodiimide. Unreacted amino groups on the agarose matrix are blocked subsequently by acetylation. One volume of YOH. ag adsorbs 75% of the ..cap alpha../sub 2/AR from 50 volumes of digitonin-solubilized preparation containing 0.2 pmol ..cap alpha../sub 2/AR/mg protein. Digitonin-solubilized preparations are derived from cholate extracts of porcine cerebral cortex containing approx. 0.075 pmol ..cap alpha../sub 2/AR/mg protein. Adsorption of ..cap alpha../sub 2/AR to YOH. ag is selective and thus is blocked by the ..cap alpha..-adrenergic antagonist phentolamine. Adsorbed ..cap alpha../sub 2/AR are eluted with 10 ..mu..M phentolamine (20% yield) after removal of non-related proteins with NaCl gradients. Following hydroxylapatite chromatography to concentrate ..cap alpha..''AR and to remove phentolamine, the ..cap alpha..AR is present at 200-400 pmol/mg protein, assayed using sub-saturating concentrations of (/sup 3/H)-yohimbine. (It is estimated that the specific activity of a homogeneous ..cap alpha../sub 2/AR preparation would be 12,000-16,000 pmol/mg protein.) The availability of large quantities of cortical ..cap alpha../sub 2/AR and a resin easily prepared from commercially-supplied reagents suggests that purification of quantities of ..cap alpha../sub 2/AR sufficient for subsequent biochemical studies is feasible.
Abstract. It has been suggested that-adren-ergic responsiveness is reduced in hypertension. To ev... more Abstract. It has been suggested that-adren-ergic responsiveness is reduced in hypertension. To eval-uate a possible alteration in human 3-receptors that might account for diminished /3-adrenergic responsiveness, we studied leukocytes from hypertensive and normotensive subjects after an overnight rest supine, and then after being ambulatory, a maneuver that increases plasma cat-echolamines approximately twofold. In supine samples, /-receptor affinity for the agonist isoproterenol was sig-nificantly reduced in hypertensives and was associated with a reduction in the proportion of/-receptors binding agonist with a high affinity from 42±6 % in normotensive subjects to 25±2 % in hypertensives (P < 0.05). Alterations in /3-adrenergic-mediated adenylate cyclase activity par-allelled the differences seen in the /-receptor affinity for agonist. In normotensive subjects, /-receptor density and the proportion of receptors binding agonist with high affinity were reciprocally correlated with ...
... Human Platelet AIpha2-Adrenergic Receptor in Membrane and Solubilized Preparations LEE E. LIM... more ... Human Platelet AIpha2-Adrenergic Receptor in Membrane and Solubilized Preparations LEE E. LIMBIRD,' JANICE L. SPECK, AND SHARON K. SMITH ... Despite the qualitatively different effects of Na on alpha2-receptor-agonist and alpha-2-receptor-antagonist inter-...
systems suggests that a common pool of GTP-binding proteins could be shared by hormonal systems c... more systems suggests that a common pool of GTP-binding proteins could be shared by hormonal systems coupled to activation and inhibition of the enzyme. Differential sensitivity to proteases (2, 3), Mn2+ (4, 5),
A Short Course on Theory and Methods, Second Edition is a primer for the study of cell surface re... more A Short Course on Theory and Methods, Second Edition is a primer for the study of cell surface receptors. The simplified discussion of methods and their underlying principles removes the usual intimidation caused by the specialized vocabulary or sophisticated mathematics that characterize many of the primary papers in this field. In this way, the basic concepts become emphasized. This volume is a starting point: a textbook as well as a manual to which the investigator can return for a refresher course, when needed.
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