NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/NCMN.1994.1009
J. Saffitz, S. Beau
{"title":"Adrenergic Receptor Autoradiography and in Situ Hybridization","authors":"J. Saffitz, S. Beau","doi":"10.1006/NCMN.1994.1009","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1009","url":null,"abstract":"Abstract The distribution of α- and β-adrenergic receptors in slices of anatomically complex tissues can be delineated autoradiographically with great precision and resolution. This brief review highlights methodological aspects of adrenergic receptor autoradiography and in situ hybridization at the light microscopic level of resolution. It focuses on technical differences between autoradiographic analysis in tissue sections and conventional radioligand binding assays in membranes prepared from tissue homogenates. it emphasizes strategies for characterizing quantitatively the distribution of specific receptor subtypes and classes using autoradiography and ways of detecting naturally occurring low-abundance adrenergic receptor mRNAs using in situ hybridization.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"17 1","pages":"76-87"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84139601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/NCMN.1994.1008
S. Baker, Malgorzata D. Deyrup
{"title":"Development of Novel Irreversible Ligands","authors":"S. Baker, Malgorzata D. Deyrup","doi":"10.1006/NCMN.1994.1008","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1008","url":null,"abstract":"Abstract Alkylating affinity labels are reactive compounds that bind receptors in a covalent manner. This property makes them useful tools with which to study the structure and function of these important biological recognition molecules. To familiarize investigators with the use of these compounds, an overview of their general properties in terms of design, pharmacology, and chemistry is presented. In addition, some experimental approaches to characterizing their irreversible effects are described. Finally, several uses of alkylating affinity labels for the determination of a receptor reserve, receptor turnover, and studies on receptor structure are briefly outlined. important limitations with the use of these compounds are discussed, and examples focusing mainly on catecholamine receptors are given.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"1 1","pages":"66-75"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84193927","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/ncmn.1994.1008
Baker Stephen P., Deyrup Malgorzata D.
{"title":"Development of Novel Irreversible Ligands","authors":"Baker Stephen P., Deyrup Malgorzata D.","doi":"10.1006/ncmn.1994.1008","DOIUrl":"https://doi.org/10.1006/ncmn.1994.1008","url":null,"abstract":"<div><p>Alkylating affinity labels are reactive compounds that bind receptors in a covalent manner. This property makes them useful tools with which to study the structure and function of these important biological recognition molecules. To familiarize investigators with the use of these compounds, an overview of their general properties in terms of design, pharmacology, and chemistry is presented. In addition, some experimental approaches to characterizing their irreversible effects are described. Finally, several uses of alkylating affinity labels for the determination of a receptor reserve, receptor turnover, and studies on receptor structure are briefly outlined. important limitations with the use of these compounds are discussed, and examples focusing mainly on catecholamine receptors are given.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"4 1","pages":"Pages 66-75"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1994.1008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72106491","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/NCMN.1994.1006
R. J. Hughes
{"title":"Adrenergic Receptors: Data and Programs on the Internet","authors":"R. J. Hughes","doi":"10.1006/NCMN.1994.1006","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1006","url":null,"abstract":"Abstract The adrenergic receptors belong to a gene superfamily whose members share a great deal of sequence homology. To date, over 60 members of this superfamily have been cloned and sequenced. Ready access to this wealth of sequence information, together with the software tools to analyze it, can facilitate experimental design and interpretation as well as expedite the realization of experimental goals. Many investigators have one or more computers connected to the Internet, often for no purpose other than to exchange electronic mail (e-mail) or to utilize the file-serving capabilities of a Novell network. These investigators are, to a large extent, unaware of the extensive resources that the Internet offers. The aim of this article is to facilitate utilization of the Internet, particularly as a tool to aid in the study of adrenergic receptors, and to guide the readers exploration of this resource. Although the Internet may at first appear to be an unfathomable morass of information, the simple command \"help\" can often be used to guide one′s path. Wherever possible, the reader is directed to retrieve the latest documentation on each topic directly from the Internet.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"46 1","pages":"41-49"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84967795","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/ncmn.1994.1002
Esbenshade Timothy A., Minneman Kenneth P.
{"title":"Adrenergic Receptor Subtypes: Pharmacological Approaches","authors":"Esbenshade Timothy A., Minneman Kenneth P.","doi":"10.1006/ncmn.1994.1002","DOIUrl":"https://doi.org/10.1006/ncmn.1994.1002","url":null,"abstract":"<div><p>Although the existence of four distinct adrenergic receptor subtypes (α<sub>1</sub>, α<sub>2</sub>, β<sub>1</sub>, β<em>2</em>) has been recognized for more than 15 years, it has recently become clear that the adrenergic receptor family is much larger than previously suspected. Development of more selective agonists and antagonists and careful comparison of pharmacological properties have led to the realization that there are nine or more adrenergic receptor subtypes. Molecular cloning of many of these subtypes, discussed in an accompanying article, supports this conclusion. The adrenergic receptors fall into three major families (α<sub>1</sub>, α<sub>2</sub>, β) based on pharmacology, structure, and signal transduction, with at least three closely related members within each family. Here, we summarize the known pharmacological differences between these receptors and evaluate the best methods currently available for distinguishing these subtypes using selective drugs.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"4 1","pages":"Pages 2-13"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1994.1002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72105685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/NCMN.1994.1003
K. Lynch, J. Harrison, W. Pearson
{"title":"Classification of Adrenergic Receptor Subtypes: Molecular Biologic Approaches","authors":"K. Lynch, J. Harrison, W. Pearson","doi":"10.1006/NCMN.1994.1003","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1003","url":null,"abstract":"Abstract Adrenergic receptors have been studied extensively for more than 30 years, first by physiological means, later with pharmacologic and biochemical approaches, and within the past several years by molecular biology. This extensive body of work provided the basis for subdividing the adrenergic receptors into β-, α 1 -, and α 2 -adrenergic receptor types and, subsequently, into β 1 -, β 2 -, α 1A -, α 1B -, α 2A -, and α 2B -adrenergic receptor subtypes. Although the pharmacologic approach indicated that there exist multiple subtypes of each type of adrenergic receptor, it was the molecular cloning of adrenergic receptor cDNAs/genes that demonstrated the existence of three genes encoding each adrenergic receptor type in humans and rats (and therefore probably in all mammals). The nine adrenergic receptor proteins expressed in cultured cells faithfully mimic the basic pharmacologic and biochemical properties ascribed to these receptors. In this article, we review the molecular cloning and characterization of the adrenergic receptors with special emphasis on the α 2 -adrenergic receptors and we discuss a classification scheme based on the hypothetical molecular evolution of the adrenergic receptors.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"18 1","pages":"14-19"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85830449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/NCMN.1994.1005
A. Strosberg
{"title":"Molecular Biology of 2-Adrenergic Receptors","authors":"A. Strosberg","doi":"10.1006/NCMN.1994.1005","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1005","url":null,"abstract":"Abstract Affinity purification of the β2-adrenergic receptor from hamster lung has led to the amino acid sequencing of a few of its peptides, followed by the molecular cloning of the corresponding cDNA. Expression of this cDNA confirmed the catecholamine binding properties of the β2-adrenergic receptor. This initial success, that of the cloning of the turkey erythrocyte β1-like and of the platelet α2A-adrenergic receptors, has rapidly led to the identification and cloning by homology of nine different subtypes of α1, α2, and β receptors. All these belong to the very large family of G-protein-coupled membrane receptors, which may include over 1000 proteins that act as receptors for neurotransmitters, hormones, and sensory signals such as light or odors. While a few of the adrenergic receptors had been characterized previously by pharmacologic means, most were actually not known to exist as individual entitles. The \"reverse pharmacology\" made possible by molecular biology should now lead to the synthesis of new subtype-selective ligands.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90209005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/NCMN.1994.1004
N. H. Lee
{"title":"Site-Directed Mutagenesis of a-Adrenergic Receptors","authors":"N. H. Lee","doi":"10.1006/NCMN.1994.1004","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1004","url":null,"abstract":"Abstract The application of in vitro site-directed mutagenesis has led to the identification of conserved amino acids that play important roles in receptor structure and function. Precise amino acid substitutions can be obtained and then correlated with changes in receptor phenotype. Here, we describe several techniques commonly employed to Introduce site-specific mutations. The benefits and potential drawbacks of each method are discussed. Site-directed mutagenesis of the human α 2A -adrenergic receptor (α 2A AR) has been successfully employed to identify conserved amino acids involved in agonist binding and receptor activation. Aspartate residues in the second and intracellular side of the third transmembrane domain of the α 2A AR are implicated in receptor/G-protein interactions. Since these aspartate residues are highly conserved among all G-protein-coupled receptors, and elimination of these residues has been shown to abolish the ability of other receptors in this class to activate their respective intracellular signaling pathways, It seems likely that these residues are critical for agonist-induced conformational changes that underlie receptor/G-protein interactions. In contrast to the role played by the conserved residues mentioned above, a conserved aspartate residue situated near the extracellular side of the third transmembrane domain plays a pivotal role in adrenergic ligand binding. Genetic analysis of the fifth transmembrane domain of the α 2A AR suggests that a conserved serine residue in this region participates in hydrogen binding to the meta -hydroxyl group of catecholamines. These findings point to the utility of site-directed mutagenesis in identifying structure-function relationships among G-protein-coupled receptors.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76594108","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/ncmn.1994.1005
Strosberg A.Donny
{"title":"Molecular Biology of β2-Adrenergic Receptors","authors":"Strosberg A.Donny","doi":"10.1006/ncmn.1994.1005","DOIUrl":"https://doi.org/10.1006/ncmn.1994.1005","url":null,"abstract":"<div><p>Affinity purification of the β<sub>2</sub>-adrenergic receptor from hamster lung has led to the amino acid sequencing of a few of its peptides, followed by the molecular cloning of the corresponding cDNA. Expression of this cDNA confirmed the catecholamine binding properties of the β<sub>2</sub>-adrenergic receptor. This initial success, that of the cloning of the turkey erythrocyte β<sub>1</sub>-like and of the platelet α<sub>2A</sub>-adrenergic receptors, has rapidly led to the identification and cloning by homology of nine different subtypes of α<sub>1</sub>, α<sub>2</sub>, and β receptors. All these belong to the very large family of G-protein-coupled membrane receptors, which may include over 1000 proteins that act as receptors for neurotransmitters, hormones, and sensory signals such as light or odors. While a few of the adrenergic receptors had been characterized previously by pharmacologic means, most were actually not known to exist as individual entitles. The \"reverse pharmacology\" made possible by molecular biology should now lead to the synthesis of new subtype-selective ligands.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"4 1","pages":"Pages 32-40"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1994.1005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72105690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NeuroprotocolsPub Date : 1994-02-01DOI: 10.1006/NCMN.1994.1010
R. J. Hughes
{"title":"Quantitative Analysis of Low-Abundance mRNA: Application to Adrenergic Receptors","authors":"R. J. Hughes","doi":"10.1006/NCMN.1994.1010","DOIUrl":"https://doi.org/10.1006/NCMN.1994.1010","url":null,"abstract":"Abstract The adrenergic receptors belong to a family of receptors that is postulated to span the plasma membrane seven times and is linked to regulatory GTP-binding proteins. These receptors mediate a wide variety of physiological responses through activation of several distinct second-messenger systems. Belying the importance of the adrenergic receptors in regulating physiological responses in target cells is their paucity. These receptors are present in very low numbers, in some cases only a thousand copies per cell. In addition, the adrenergic receptors are relatively stable proteins. Thus, cells have no need to synthesize large amounts of these proteins and, in consequence, the level of mRNA coding for these receptors is very low. In this paper, I examine some of the ways in which quantitation of these scarce mRNA species has been approached, with particular emphasis on the use of polymerase chain reaction.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"6 1","pages":"88-94"},"PeriodicalIF":0.0,"publicationDate":"1994-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73355744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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