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11.
Madan M. Kwatra Jolanda Schreurs Debra A. Schwinn Michael A. Innis Marc G. Caron Robert J. Lefkowitz 《Protein expression and purification》1995,6(6)
To obtain large quantities of pure human β2-adrenergic receptor (β2-AR) needed for structural studies, an efficient method for β2-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged β2-AR was expressed in Sf9 cells with a specific activity of 5–20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-β-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60–70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of >30%. The purified receptor was concentrated to >1 mg/ml without loss of ligand binding activity. 相似文献
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14.
Gary L. Stiles Ruth H. Strasser Brian F. Kilpatrick Sabrina R. Taylor Robert J. Lefkowitz 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,802(3)
Photoaffinity labeling techniques have recently demonstrated that mammalian β1- and β2-adrenergic receptors reside on peptides of Mr 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of Mr 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β2-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of Mr 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [3H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[125I]iodobenzylcarazolol. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein. 相似文献
15.
The beta 1-adrenergic receptor of the turkey erythrocyte. Molecular heterogeneity revealed by purification and photoaffinity labeling 总被引:6,自引:0,他引:6
R G Shorr M W Strohsacker T N Lavin R J Lefkowitz M G Caron 《The Journal of biological chemistry》1982,257(20):12341-12350
The beta 1-adrenergic receptor of turkey erythrocytes has been purified by a combination of affinity and high performance steric exclusion chromatography. These procedures provide preparations with specific activities of greater than 15,000 pmol/mg of protein with an overall recovery of approximately 30% of the receptor activity solubilized from membrane preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated purified receptor reveals two bands of labeled protein with apparent Mr = 40,000 +/- 2,000 and 45,000 +/- 3,000 in a 3-4:1 ratio. These same two peptides can also be labeled specifically and in approximately the same ration in both membranes and purified preparations using the photoaffinity probe 125I-labeled p-azidobenzylcarazolol. When the two purified polypeptides are completely separated by high performance liquid chromatography and subjected to detailed ligand binding studies, identical beta 1-adrenergic specificities are found for the two receptor forms. Preliminary characterization of these two proteins by partial protease digestion suggests a large degree of similarity between them, albeit with some significant differences. These results demonstrate that both purification and photoaffinity labeling identify two polypeptides in turkey erythrocyte membranes as containing a beta 1-adrenergic receptor binding site. The functional and structural relationships of these two forms of the receptor remain to be elucidated. 相似文献
16.
S B Liggett J Ostrowski L C Chesnut H Kurose J R Raymond M G Caron R J Lefkowitz 《The Journal of biological chemistry》1992,267(7):4740-4746
To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites. 相似文献
17.
M A Kjelsberg S Cotecchia J Ostrowski M G Caron R J Lefkowitz 《The Journal of biological chemistry》1992,267(3):1430-1433
Mutations in an intracellular region of the alpha 1B-adrenergic receptor constitutively activate the receptor, resulting in G protein coupling in the absence of agonist, as evidenced by elevated levels of polyphosphoinositide hydrolysis. Remarkably, all 19 possible amino acid substitutions at a single site in this region (alanine 293) confer constitutive activity. This set of mutated receptors exhibits a graded range of elevated biological activities, apparently representing a spectrum of receptor conformations which mimic the "active" state of the wild type receptor. In addition to their constitutive activities, these mutated receptors all demonstrate a higher affinity for agonists, another primary characteristic of the "active" conformation of G protein-coupled receptors. The fact that all possible mutations at this particular site result in increased activity suggests that this region may function to constrain the G protein coupling of the receptor, a constraint which is normally relieved by agonist occupancy. 相似文献
18.
Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor 总被引:8,自引:0,他引:8
Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the beta 2-adrenergic receptor (beta AR). The presence of disulfide bonds at the ligand binding site is indicated by "competitive" inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by beta-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, we indeed were able to identify four cysteines which are critical for normal ligand binding affinities and for the proper expression of functional beta AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that, in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site. 相似文献
19.
Molecular cloning and expression of the cDNA for a novel alpha 1-adrenergic receptor subtype 总被引:27,自引:0,他引:27
D A Schwinn J W Lomasney W Lorenz P J Szklut R T Fremeau T L Yang-Feng M G Caron R J Lefkowitz S Cotecchia 《The Journal of biological chemistry》1990,265(14):8183-8189
A novel alpha 1-adrenergic receptor subtype has been cloned from a bovine brain cDNA library. The deduced amino acid sequence is that of a 466-residue polypeptide. The structure is similar to that of the other adrenergic receptors as well as the larger family of G protein-coupled receptors that have a presumed seven-membrane-spanning domain topography. The greatest sequence identity of this receptor protein is with the previously cloned hamster alpha 1B-adrenergic receptor being approximately 72% within the presumed membrane-spanning domains. Localization on different human chromosomes provides evidence that the bovine cDNA is distinct from the hamster alpha 1B-adrenergic receptor. The bovine cDNA clone expressed in COS7 cells revealed 10-fold higher affinity for the alpha 1-adrenergic antagonists WB4101 and phentolamine and the agonist oxymetazoline as compared with the alpha 1B receptor, results similar to pharmacologic binding properties described for the alpha 1A receptor. Despite these similarities in pharmacological profiles, the bovine alpha 1-adrenergic receptor is sensitive to inhibition by the alkylating agent chloroethylclonidine unlike the alpha 1A-adrenergic receptor subtype. In addition, a lack of expression in tissues where the alpha 1A subtype exists suggests that this receptor may actually represent a novel alpha 1-adrenergic receptor subtype not previously appreciated by pharmacological criteria. 相似文献
20.
We have investigated alterations in beta-adrenergic receptors in rat myocardial membranes derived from hypothyroid and hyperthyroid animals. (-)Isoproterenol competition curves with (-)[3H]dihydroalprenolol revealed that isoproterenol binds to the beta-adrenergic receptor with two distinct affinity states having high (KH) and low (KL) dissociation constants. In the presence of guanine nucleotides the isoproterenol competition curve steepened and had a higher EC50 (50% displacement). This was due to a transition of the high affinity state to a uniformly low affinity state. Using computer modeling of these competition curves, we have demonstrated that in hyperthyroidism, the isoproterenol curve in the absence of guanine nucleotides is shifted to the left with the EC50 changing from 180 ± 40 to 80 ± 20 nM (p < .02). The fold shift (4 fold) in KH (nM) 30 ± 9 to 7 ± 2 (p < .001) is greater than that (1.6 fold) in KL (nM) 595 ± 56 to 376 ± 34 (p < .001) such that the KL/KH ratio shifted from 20 ± 3 to 54 ± 9 (p < .001). The ratio, KL/KH, for a particular agonist appears to be related to its efficacy in activating adenylate cyclase.There was no significant alteration in any of these parameters in hypothyroid animals. Receptor number was decreased in hypothyroidism, 16 ± 3 fmol/mg protein (p < .03) and increased in hyperthyroidism 44 ± 4 (p < .03) compared to control 26 ± 2.In the rat heart agonist affinity and receptor number are modulated in hyperthyroidism, but only receptor number in hypothryoidism. Thus thyroid hormone can modify not only receptor number but agonist affinity as well. 相似文献