adrenergic receptor protocols

Edited by Curtis A. Machida Methods in Molecular Biology TM VOLUME 126 HUMANA PRESS Adrenergic Receptor Protocols HUMANA PRESS Methods in Molecular Biology TM Adrenergic Receptor Protocols Edited by Curtis A. Machida Library Construction 3 3 From: Methods in Molecular Biology, vol. 126: Adrenergic Receptor Protocols Edited by: C. A. Machida © Humana Press Inc., Totowa, NJ 1 Construction of Libraries for Isolation of Adrenergic Receptor Genes Margaret A. Scofield, Jean D. Deupree, and David B. Bylund 1. Introduction 1.1. Adrenergic Receptors Adrenergic receptors mediate the central and peripheral actions of norepi- nephrine and epinephrine. Both of these catecholamine messengers play important roles in the regulation of diverse physiological systems and are widely distributed throughout the body. Agonists and antagonists interacting with adrenergic receptors have proven useful in the treatment of a variety of cardiovascular, respiratory, and mental disorders (1,2). Adrenergic receptors were originally divided into two major types, α-adren- ergic receptor (α-AR) and β-adrenergic receptor (β-AR), based on their phar- macological characteristics (i.e., rank order potency of agonists) (3). Subsequently, the α-AR and β-AR types were further subdivided into α 1 -AR, α 2 -AR, β 1 -AR, and β 2 -AR subtypes (for a more complete historical perspec- tive see refs. 4,5). Based on both pharmacological and molecular evidence, it is now clear that a more useful classification scheme is based on three major types—α 1 -AR, α 2 -AR, and β-AR—each of which is further divided into three or four subtypes (Fig. 1) (4). 1.1.1. α 1 -AR Subtypes α 1A -AR and α 1B -AR subtypes were defined pharmacologically based on the differential affinities of WB 4101 and phentolamine (6–8), and on selective receptor inactivation by the alkylating agent chlorethylclonidine. Three α 1 -AR subtypes have been identified by molecular cloning (Table 1). The α 1B -AR from hamster was cloned first (9), followed by the bovine α 1A -AR, which 4 Scofield, Deupree, and Bylund unfortunately was prematurely identified as the α 1C -AR subtype (10,11). The α 1D -AR was cloned from the rat (12,13), although this receptor was prema- turely called the α 1A -AR. A fourth α 1 -AR subtype, called the α 1L -(based on its low affinity for prazosin), has been suggested (14,15), although its exist- ence is debatable (16). 1.1.2. α 2 -AR Subtypes The evidence for α 2 -AR subtypes initially came from binding and func- tional studies in various tissues and cell lines (17). On the basis of these stud- ies, three genetic and four pharmacological α 2 -AR subtypes have been defined. The α 2A -AR and α 2B -AR subtypes were initially defined based on differential affinity for adrenergic agents, such as prazosin and oxymetazoline (4). These subtypes were subsequently cloned from a variety of species (Table 2). A third subtype, α 2C -AR, was identified originally in an opossum kidney cell line (18,19) and has also been cloned from several species (Table 2). A fourth pharmacological subtype, the α 2D -AR, has been identified in the rat and cow (20,21). This pharmacological subtype is a species ortholog of the human α 2A -AR subtype and, thus, is not considered to be a separate genetic subtype. 1.1.3. β -AR Subtypes The β 1 -AR and β 2 -AR subtypes were identified as early as 1967 based on a comparison of the rank orders of potency of a variety of agonists (22). Highly selective antagonists for both β 1 -AR and β 2 -AR have been subsequently developed. More recently, it became apparent that not all of the β-AR-mediated responses can be classified as either β 1 -AR or β 2 -AR, and thus, the β 3 -AR was identified (23,24). This receptor has low affinity for the commonly used β antagonists and has often been referred to as the “atypical” β-AR. These Fig. 1. The current classification scheme for adrenergic receptors. (text continued on page 13) β-AR α 2 -AR α 1 -AR β 1 -AR β 2 -AR β 3 -AR β 4 -AR α 1A -AR α 1B -AR α 1D -AR α 2A -AR α 2B -AR α 2C -AR Library Construction 5 5 Table 1 α 1 -Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomic b Flanking nucleotides, subtype a common name identification in text number tissu e type region coding sequence α 1A -AR Oryctolagus cuniculus α 1a -adrenoceptor U81982 cDNA (c) No 1401 bp rabbit liver (1 1401) O. cuniculus α 1c -adrenoceptor subtype S75999 cDNA (p) No 432 bp rabbit brain (1 430) Homo sapiens adrenergic α 1c receptor U03866 cDNA (c) No 1500 bp human prostate (66 1466) H. sapiens α 1C -AR L31774 cDNA (c) No 1401 bp human prostate (1 1401) H. sapiens α 1C -AR D25235 cDNA (c) No 2290 bp human prostate (437 1837) H. sapiens α 1C -AR U02569 cDNA (c) No 1902 bp human prostate (425 1825) H. sapiens α 1A -AR AF013261 cDNA (c) No 1765 bp human isoform 4 prostate (201 1568) H. sapiens α 1C -adrenoceptor receptor D32201 cDNA (c) No 2089 bp human isoform 3 prostate (437 1726) H. sapiens α 1C -AR D32202 cDNA (c) No 2306 bp human isoform 2 prostate (437 1936) H. sapiens α 1c -adrenoceptor subtype S76001 cDNA (p) No 432 bp human brain, (1 431) saphenous vein (continued) 6 Scofield, Deupree, and Bylund Table 1 (continued) α 1 -Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomic b Flanking nucleotides, subtype a common name identification in text number tissu e type region coding sequence H. sapiens α 1a -AR U72653 Genomic Yes 6195 bp human (no coding region) Bos taurus α 1C -AR J05426 cDNA (c) No 2461 bp cow adult brain (97 1497) cortex Mus musculus α 1a -AR S80220 cDNA (p) No 252 bp house mouse brain (1 252) M. musculus α 1A -AR AF031431 cDNA (c) No 1401 bp house mouse brain, liver, (1 1401) kidney Rattus norvegicus α 1C -AR U13368 cDNA (c) No 1862 bp Norway rat cardiac (36 1436) myocytes R. norvegicus α 1c -AR U07126 cDNA (c) No 1428 bp Norway rat brain (16 1416) Meriones unguiculatus α 1a -AR AF047188 cDNA (p) No 175 bp Mongolian gerbil spiral modiolar (1 175) artery Oryzias latipes α 1A adrenoceptor D63859 Genomic (c) No 1770 bp Japanese medaka fish (228 1640) α 1B -AR M. musculus α 1B -AR Y12738 cDNA (c) No 2268 bp house mouse brain (724 2268) 6 Library Construction 7 Mus. musculus α 1b -AR S80219 cDNA (p) No 120 bp house mouse brain (3 19) R. norvegicus α 1B -AR X51585 cDNA (c) No 2086 bp Norway rat brain (241 1788) R. norvegicus α 1B -AR M60655 cDNA (c) No 2108 bp Norway rat brain (15 1562) R. norvegicus α 1B -AR D32045 Genomic Yes 2387 bp Norway rat (1628 2387) R. norvegicus α 1B -AR U83985 Genomic Yes 513 bp Norway rat (no coding region) R. norvegicus α 1B -AR L08609 Genomic (p) Yes 2207 bp Norway rat exon 1 (1209 2157, L08610:51 649) R. norvegicus α 1B -AR L08610 Genomic (p) No 1365 bp Norway rat exon 2 (L08609: 1209 2157, 51 649) M. unguiculatus α 1b -AR AF047189 cDNA (p) No 258 bp Mongolian gerbil spiral modiolar artery (1 258) Mesocricetus auratus α 1B -AR J04084 cDNA (c) No 2089 bp Syrian golden hamster smooth muscle (15 1562) H. sapiens adrenergic α 1b receptor U03865 cDNA (c) No 1738 bp human brainstem (124 1686) H. sapiens α 1B -AR L31773 cDNA (c) No 1560 bp human heart (1 1560) H. sapiens α 1B -AR M99589 Genomic Yes 2669 bp human (no coding region) (continued) 7 8 Scofield, Deupree, and Bylund Table 1 (continued) α 1 -Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomic b Flanking nucleotides, subtype a common name identification in text number tissu e type region coding sequence Canis familiaris RDC5 mRNA for G protein- X14050 cDNA (p) No 1695 bp dog coupled receptor thyroid (1 1256) α 1D -AR R. norvegicus α 1A -AR M60654 cDNA (c) No 2936 bp Norway rat brain (480 2162) R. norvegicus α 1a/d -AR L31771 cDNA (c) No 2939 bp Norway rat cerebral cortex (480 2165) R. norvegicus α 1D -AR AF071014 Genomic (p) Yes 1783 bp Norway rat (1597 1783) M. musculus α 1d -AR S80044 cDNA (c) No 1902 bp house mouse brain (118 1806) M. musculus α 1A -AR L20333 cDNA (p) No 483 bp house mouse homolog testis (1 483) H. sapiens α 1a/d -AR L31772 Genomic and No 1831 bp human cDNA (c) (1 1719) prostate H. sapiens adrenergic α 1a receptor U03864 cDNA (c) No 1860 bp human hippocampus (58 1776) H. sapiens α 1A/D -AR D29952 Genomic and No 2077 bp human cDNA (c) (5 1723) prostate H. sapiens α A1 -AR M76446 cDNA (c) No 2002 bp human brain (56 1561) O. cuniculus α 1d adrenoceptor U64032 cDNA (c) No 1731 bp rabbit liver (1 1731) a Assignment of subtype is based on the alignment and groupings of coding sequences generated by the denogram of the program Pile up, GCG. b (c) Complete coding sequence; (p) partial coding sequence. 8 Library Construction 9 Table 2 α 2 -Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomic b Flanking nucleotides, subtype a common name identification in text number tissu e type region coding sequence α 2A -AR Rattus rattus α 2D -AR U79031 cDNA (c) No 1552 bp black rat brain (1 1353) R. norvegicus α 2 -AR-RG20 M62372 Genomic (c) No 1380 bp Norway rat (1 1353) R. norvegicus α 2D -AR U49747 Genomic (p) Yes 2836 bp Norway rat (2831 2836) M. musculus α 2 -AR M99377 Genomic (c) No 1454 bp house mouse (α 2 -AR-C10 homolog) (51 1403) M. musculus α 2A -AR U29693 Genomic Yes 2828 bp house mouse (no coding region) M. auratus α 2 receptor adrenergic L28124 cDNA (p) No 313 bp golden hamster (α 2A -AR) adipocytes (1 313) Sus scrofa α 2A -AR J05652 Genomic (c) No 1728 bp pig (α 2 -AR-C10 homolog) (130 1482) Bos taurus α 2D -AR U79030 Genomic (c) Yes 2923 bp cow (1509 2867) B. taurus adrenergic receptor S66295 cDNA (p) No 120 bp cow subtype α 2D -AR pineal gland (1 120) H. sapiens α 2 -AR M18415 Genomic (c) No 1521 bp human (59 1411) H. sapiens α 2 -AR M23533 Genomic (c) Yes 3604 bp human (α-2A) (2078 3430) Gallus gallus adrenergic receptor S66185 Genomic (p) No 117 bp chicken subtype α 2A -AR (1 117) (continued) 9 10 Scofield, Deupree, and Bylund Table 2 (continued) α 2 -Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomic b Flanking nucleotides, subtype a common name identification in text number tissu e type region coding sequence Cavia porcellus α 2A -AR adrenoceptor U25722 Genomic (c) No 2291 bp guinea pig (49 1401) α 2B -AR R. norvegicus α 2B -AR M32061 cDNA (c) No 2319 bp Norway rat (RNG-α 2 -AR) kidney (366 1727) R. norvegicus α 2B -AR X74400 Genomic (c) No 1639 bp Norway rat (178 1524) M. musculus α 2 -AR L00979 Genomic (c) No 1650 bp house mouse (α 2C2 -AR homolog) (227 1573) M. musculus α 2 -AR M94583 Genomic (c) Yes 5265 bp house mouse (α 2C2 -AR homolog) (1146 2513) Elephas maximus α 2B -AR Y12525 Genomic (p) No 1153 bp Indian elephant (1 1153) Dugong dugon α-AR Y15947 Genomic (p) No 1171 bp sea cow subtype 2B (1 1171) Procavia capensis α 2B -AR Y12523 Genomic (p) No 1168 bp cape rock hyrax (1 1168) (shrewmouse) Orycteropus afer α 2B -AR Y12522 Genomic (p) No 1165 bp aardvark (1 1165) Amblysomus hottentotus α 2B -AR Y12526 Genomic (p) No 1159 bp golden moles (1 1159) Echinops telfairi α-AR Y17692 Genomic (p) No 1153 bp Madagascar hedgehog subtype 2B (1 1153) Macroscelides α 2B -AR Y12524 Genomic (p) No 1162 bp proboscideus (1 1162) short-eared elephant shrew 10 Library Construction 11 B. taurus α-AR Y15944 Genomic (p) No 1177 bp cow subtype 2B (1 1177) Equus caballus α-AR Y15945 Genomic (p) No 1168 bp horse subtype 2B (1 1168) Erinaceus europaeus α 2B -AR Y12521 Genomic (p) No 1174 bp western European (1 1174) hedgehog Talpa europaea α 2B -AR Y12520 Genomic (p) No 1192 bp European mole (1 1192) H. sapiens α 2B -AR AF005900 Genomic (c) Yes 9842 bp human (α 2C2 -AR) (5398 6750) H. sapiens α 2 -AR M34041 Genomic (c) No 2072 bp human (α 2 -AR c2) (413 1765) H. sapiens α 2 -AR-ADRA2C M38742 Genomic (p) No 885 bp human (1 885) O. cuniculus α-AR Y15946 Genomic (p) No 1183 bp rabbit subtype 2B (1 1183) C. porcellus α 2B adrenoceptor gene U25723 Genomic (c) No 1987 bp guinea pig (328 1674) Didelphis marsupialis α-AR Y15943 Genomic (p) No 1147 bp opossum subtype 2B (1 1147) α 2C -AR M. musculus α 2 -AR M97516 Genomic (c) No 2409 bp house mouse (α 2 -C4 homolog) (415 1791) M. musculus α 2 -AR M99376 Genomic (c) No 1503 bp house mouse (α 2 -C4 homolog) (51 1427) R. norvegicus α 2 -AR-RG10 M62371 Genomic (c) No 1380 bp Norway rat (1 1377) R. norvegicus α 2 -C4-AR X57659 Genomic and ? 2991 bp Norway rat cDNA (c) (907 2283) brain (continued) 11 [...]... laevis African clawed frog Meleagris gallopavo turkey β1-AR Y09213 adrenergic β4c receptor U13977 β1-AR like M gallopavo turkey adrenergic β4c receptor U13978 cDNA (c) fetal red blood cells cDNA (c) embryo cDNA (c) fetal red blood cells Genomic (c) β2-AR B taurus cow B taurus cow S scrofa pig S scrofa pig β2-AR Z86037 adrenergic β2 receptor Library Construction 15 R norvegicus Norway rat R norvegicus... Differentiation of α1 -adrenergic receptors linked to phosphatidylinositol turnover and cyclic AMP accumulation in rat brain Mol Pharmacol 31, 239–246 7 Minneman, K P., Han, C., and Abel, P W (1988) Comparison of α1 -adrenergic receptor subtypes distinguished by chloroethylclonidine and WB4101 Mol Pharmacol 33, 509–514 8 Morrow, A L and Creese, I (1986) Characterization of α1 -adrenergic receptor subtypes... α2 -adrenergic receptors FASEB J 6, 832–839 18 Blaxall, H S., Murphy, T J., Baker, J C., Ray, C., and Bylund, D B (1991) Library Construction 19 20 21 22 23 24 25 26 27 28 29 30 31 33 Characterization of the alpha-2C adrenergic receptor subtype in the opossum kidney and in the OK cell line J Pharmacol Exp Ther 259, 323–329 Murphy, T J and Bylund, D B (1988) Characterization of alpha-2 adrenergic receptors... in small frozen aliquots that are then discarded after having been opened several times Listed below are stock solutions that are used in many molecular biology protocols (text continued on page 20) 14 Table 3 β -Adrenergic Receptors Adrenergic receptor subtypea β1-AR Species, common name H sapiens human H sapiens human M unguiculatus Mongolian gerbil Definition in GenBank database, subtype identification... sapiens human H sapiens human H sapiens human H sapiens human H sapiens human M mulatta rhesus monkey C familiaris dog C familiaris dog Felis catus cat Rattus sp rat 18 Table 3 (continued) β -Adrenergic Receptors Adrenergic receptor subtypea Species, common name Definition in GenBank database, subtype identification in text Accession number cDNA or genomicb tissue type 5′ Flanking Number of nucleotides, region.. .Adrenergic receptor subtypea Species, common name 12 Table 2 (continued) α2 -Adrenergic Receptors Definition in GenBank database, subtype identification in text 12 cDNA or genomicb tissue type D00819 Genomic (c) No M58316 cDNA (c) brain cDNA (c)... α1 -adrenergic receptor Proc Natl Acad Sci USA 85, 7159–7163 10 Ford, A P D W., Williams, T J., Blue, D R., and Clarke, D E (1994) α1-Adrenoceptor classification: sharpening Occam’s razor Trends Pharmacol Sci 15, 167–170 11 Schwinn, D A., Lomasney, J W., Lorenz, W., Szklut, P J., Fremeau, R T., Jr., Yang-Feng, T L., et al (1990) Molecular cloning and expression of the cDNA for a novel α1 -adrenergic receptor. .. L., et al (1991) Molecular cloning and expression of the cDNA for the α1A -adrenergic receptor: the gene for which is located on human chromosome 5 J Biol Chem 266, 6365–6369 13 Perez, D M., Piascik, M T., and Graham, R M (1991) Solution-phase library screening for the identification of rare clones: isolation of an α1D -adrenergic receptor cDNA Mol Pharmacol 40, 876–883 14 Muramatsu, I., Ohmura, T., Hashimoto,... (c) Yes No 1584 bp (301 1458) 1533 bp (89 1375) 3445 bp (892 2138, 2548 2587) 2032 bp (224 1480) 376 bp (1 376) 329 bp (1 329) 5288 bp (1758 3014) (continued) 15 16 Table 3 (continued) β -Adrenergic Receptors Adrenergic receptor subtypea Species, common name 16 Accession number cDNA or genomicb tissue type β2-AR X17607 Genomic (c) Yes β2-AR L39264 Genomic (c) Yes β2-AR J03024 No β2-AR U35448 cDNA (c) heart... Brent, R., Kingston, R E., Moore, D D., Seidman, J G., Smith, J A., et al (1988) Plating lambda phage to generate plaques, in Current Protocols in Molecular Biology, vol 1, Wiley, Interscience, New York, pp 1.11.1.–1.11.4 Receptor Gene Isolation 37 2 Isolation of Adrenergic Receptor Genes Margaret A Scofield, Jean D Deupree, and David B Bylund 1 Introduction In order to isolate a single gene, phage or . in Molecular Biology TM VOLUME 126 HUMANA PRESS Adrenergic Receptor Protocols HUMANA PRESS Methods in Molecular Biology TM Adrenergic Receptor Protocols Edited by Curtis A. Machida Library Construction. Molecular Biology, vol. 126: Adrenergic Receptor Protocols Edited by: C. A. Machida © Humana Press Inc., Totowa, NJ 1 Construction of Libraries for Isolation of Adrenergic Receptor Genes Margaret. gallus adrenergic receptor S66185 Genomic (p) No 117 bp chicken subtype α 2A -AR (1 117) (continued) 9 10 Scofield, Deupree, and Bylund Table 2 (continued) α 2 -Adrenergic Receptors Adrenergic Definition