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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 α1-AR, α2-AR, β1-AR, and β2-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—α1-AR, α2-AR, and β-AR—each of which is further divided into three or four subtypes (Fig. 1) (4). 1.1.1. α1-AR Subtypes α1A-AR and α1B-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 α1-AR subtypes have been identified by molecular cloning (Table 1). The α1B-AR from hamster was cloned first (9), followed by the bovine α1A-AR, which 4 Scofield, Deupree, and Bylund unfortunately was prematurely identified as the α1C-AR subtype (10,11). The α1D-AR was cloned from the rat (12,13), although this receptor was prema- turely called the α1A-AR. A fourth α1-AR subtype, called the α1L-(based on its low affinity for prazosin), has been suggested (14,15), although its exist- ence is debatable (16). 1.1.2. α2-AR Subtypes The evidence for α2-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 α2-AR subtypes have been defined. The α2A-AR and α2B-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, α2C-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 α2D-AR, has been identified in the rat and cow (20,21). This pharmacological subtype is a species ortholog of the human α2A-AR subtype and, thus, is not considered to be a separate genetic subtype. 1.1.3. β-AR Subtypes The β1-AR and β2-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 β1-AR and β2-AR have been subsequently developed. More recently, it became apparent that not all of the β-AR-mediated responses can be classified as either β1-AR or β2-AR, and thus, the β3-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 α2-AR α1-AR β1-AR β2-AR β3-AR β4-AR α1A-AR α1B-AR α1D-AR α2A-AR α2B-AR α2C-AR Library Construction 5 5 Table 1 α1-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence α1A-AR Oryctolagus cuniculus α1a-adrenoceptor U81982 cDNA (c) No 1401 bp rabbit liver (1..1401) O. cuniculus α1c-adrenoceptor subtype S75999 cDNA (p) No 432 bp rabbit brain (1..430) Homo sapiens adrenergic α1c receptor U03866 cDNA (c) No 1500 bp human prostate (66..1466) H. sapiens α1C-AR L31774 cDNA (c) No 1401 bp human prostate (1..1401) H. sapiens α1C-AR D25235 cDNA (c) No 2290 bp human prostate (437..1837) H. sapiens α1C-AR U02569 cDNA (c) No 1902 bp human prostate (425..1825) H. sapiens α1A-AR AF013261 cDNA (c) No 1765 bp human isoform 4 prostate (201..1568) H. sapiens α1C-adrenoceptor receptor D32201 cDNA (c) No 2089 bp human isoform 3 prostate (437..1726) H. sapiens α1C-AR D32202 cDNA (c) No 2306 bp human isoform 2 prostate (437..1936) H. sapiens α1c-adrenoceptor subtype S76001 cDNA (p) No 432 bp human brain, (1..431) saphenous vein (continued) 6 Scofield, Deupree, and Bylund Table 1 (continued) α1-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence H. sapiens α1a-AR U72653 Genomic Yes 6195 bp human (no coding region) Bos taurus α1C-AR J05426 cDNA (c) No 2461 bp cow adult brain (97..1497) cortex Mus musculus α1a-AR S80220 cDNA (p) No 252 bp house mouse brain (1..252) M. musculus α1A-AR AF031431 cDNA (c) No 1401 bp house mouse brain, liver, (1..1401) kidney Rattus norvegicus α1C-AR U13368 cDNA (c) No 1862 bp Norway rat cardiac (36..1436) myocytes R. norvegicus α1c-AR U07126 cDNA (c) No 1428 bp Norway rat brain (16..1416) Meriones unguiculatus α1a-AR AF047188 cDNA (p) No 175 bp Mongolian gerbil spiral modiolar (1..175) artery Oryzias latipes α1A adrenoceptor D63859 Genomic (c) No 1770 bp Japanese medaka fish (228..1640) α1B-AR M. musculus α1B-AR Y12738 cDNA (c) No 2268 bp house mouse brain (724..2268) 6 Library Construction 7 Mus. musculus α1b-AR S80219 cDNA (p) No 120 bp house mouse brain (3..19) R. norvegicus α1B-AR X51585 cDNA (c) No 2086 bp Norway rat brain (241..1788) R. norvegicus α1B-AR M60655 cDNA (c) No 2108 bp Norway rat brain (15..1562) R. norvegicus α1B-AR D32045 Genomic Yes 2387 bp Norway rat (1628..2387) R. norvegicus α1B-AR U83985 Genomic Yes 513 bp Norway rat (no coding region) R. norvegicus α1B-AR L08609 Genomic (p) Yes 2207 bp Norway rat exon 1 (1209..2157, L08610:51..649) R. norvegicus α1B-AR L08610 Genomic (p) No 1365 bp Norway rat exon 2 (L08609: 1209..2157, 51..649) M. unguiculatus α1b-AR AF047189 cDNA (p) No 258 bp Mongolian gerbil spiral modiolar artery (1..258) Mesocricetus auratus α1B-AR J04084 cDNA (c) No 2089 bp Syrian golden hamster smooth muscle (15..1562) H. sapiens adrenergic α1b receptor U03865 cDNA (c) No 1738 bp human brainstem (124..1686) H. sapiens α1B-AR L31773 cDNA (c) No 1560 bp human heart (1..1560) H. sapiens α1B-AR M99589 Genomic Yes 2669 bp human (no coding region) (continued) 7 8 Scofield, Deupree, and Bylund Table 1 (continued) α1-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence Canis familiaris RDC5 mRNA for G protein- X14050 cDNA (p) No 1695 bp dog coupled receptor thyroid (1..1256) α1D-AR R. norvegicus α1A-AR M60654 cDNA (c) No 2936 bp Norway rat brain (480..2162) R. norvegicus α1a/d-AR L31771 cDNA (c) No 2939 bp Norway rat cerebral cortex (480..2165) R. norvegicus α1D-AR AF071014 Genomic (p) Yes 1783 bp Norway rat (1597..1783) M. musculus α1d-AR S80044 cDNA (c) No 1902 bp house mouse brain (118..1806) M. musculus α1A-AR L20333 cDNA (p) No 483 bp house mouse homolog testis (1..483) H. sapiens α1a/d-AR L31772 Genomic and No 1831 bp human cDNA (c) (1..1719) prostate H. sapiens adrenergic α1a receptor U03864 cDNA (c) No 1860 bp human hippocampus (58..1776) H. sapiens α1A/D-AR D29952 Genomic and No 2077 bp human cDNA (c) (5..1723) prostate H. sapiens αA1-AR M76446 cDNA (c) No 2002 bp human brain (56..1561) O. cuniculus α1d adrenoceptor U64032 cDNA (c) No 1731 bp rabbit liver (1..1731) aAssignment of subtype is based on the alignment and groupings of coding sequences generated by the denogram of the program Pileup, GCG. b(c) Complete coding sequence; (p) partial coding sequence. 8 Library Construction 9 Table 2 α2-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence α2A-AR Rattus rattus α2D-AR U79031 cDNA (c) No 1552 bp black rat brain (1..1353) R. norvegicus α2-AR-RG20 M62372 Genomic (c) No 1380 bp Norway rat (1..1353) R. norvegicus α2D-AR U49747 Genomic (p) Yes 2836 bp Norway rat (2831..2836) M. musculus α2-AR M99377 Genomic (c) No 1454 bp house mouse (α2-AR-C10 homolog) (51..1403) M. musculus α2A-AR U29693 Genomic Yes 2828 bp house mouse (no coding region) M. auratus α2 receptor adrenergic L28124 cDNA (p) No 313 bp golden hamster (α2A-AR) adipocytes (1..313) Sus scrofa α2A-AR J05652 Genomic (c) No 1728 bp pig (α2-AR-C10 homolog) (130..1482) Bos taurus α2D-AR U79030 Genomic (c) Yes 2923 bp cow (1509..2867) B. taurus adrenergic receptor S66295 cDNA (p) No 120 bp cow subtype α2D-AR pineal gland (1..120) H. sapiens α2-AR M18415 Genomic (c) No 1521 bp human (59..1411) H. sapiens α2-AR M23533 Genomic (c) Yes 3604 bp human (α-2A) (2078..3430) Gallus gallus adrenergic receptor S66185 Genomic (p) No 117 bp chicken subtype α2A-AR (1..117) (continued) 9 10 Scofield, Deupree, and Bylund Table 2 (continued) α2-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence Cavia porcellus α2A-AR adrenoceptor U25722 Genomic (c) No 2291 bp guinea pig (49..1401) α2B-AR R. norvegicus α2B-AR M32061 cDNA (c) No 2319 bp Norway rat (RNG-α2-AR) kidney (366..1727) R. norvegicus α2B-AR X74400 Genomic (c) No 1639 bp Norway rat (178..1524) M. musculus α2-AR L00979 Genomic (c) No 1650 bp house mouse (α2C2-AR homolog) (227..1573) M. musculus α2-AR M94583 Genomic (c) Yes 5265 bp house mouse (α2C2-AR homolog) (1146..2513) Elephas maximus α2B-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 α2B-AR Y12523 Genomic (p) No 1168 bp cape rock hyrax (1..1168) (shrewmouse) Orycteropus afer α2B-AR Y12522 Genomic (p) No 1165 bp aardvark (1..1165) Amblysomus hottentotus α2B-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 α2B-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 α2B-AR Y12521 Genomic (p) No 1174 bp western European (1..1174) hedgehog Talpa europaea α2B-AR Y12520 Genomic (p) No 1192 bp European mole (1..1192) H. sapiens α2B-AR AF005900 Genomic (c) Yes 9842 bp human (α2C2-AR) (5398..6750) H. sapiens α2-AR M34041 Genomic (c) No 2072 bp human (α2-AR c2) (413..1765) H. sapiens α2-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 α2B 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) α2C-AR M. musculus α2-AR M97516 Genomic (c) No 2409 bp house mouse (α2-C4 homolog) (415..1791) M. musculus α2-AR M99376 Genomic (c) No 1503 bp house mouse (α2-C4 homolog) (51..1427) R. norvegicus α2-AR-RG10 M62371 Genomic (c) No 1380 bp Norway rat (1..1377) R. norvegicus α2-C4-AR X57659 Genomic and ? 2991 bp Norway rat cDNA (c) (907..2283) brain (continued) 11

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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 α1-AR, α2-AR, β1-AR, and β2-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—α1-AR, α2-AR, and β-AR—each of which is further divided into three or four subtypes (Fig. 1) (4). 1.1.1. α1-AR Subtypes α1A-AR and α1B-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 α1-AR subtypes have been identified by molecular cloning (Table 1). The α1B-AR from hamster was cloned first (9), followed by the bovine α1A-AR, which 4 Scofield, Deupree, and Bylund unfortunately was prematurely identified as the α1C-AR subtype (10,11). The α1D-AR was cloned from the rat (12,13), although this receptor was prema- turely called the α1A-AR. A fourth α1-AR subtype, called the α1L-(based on its low affinity for prazosin), has been suggested (14,15), although its exist- ence is debatable (16). 1.1.2. α2-AR Subtypes The evidence for α2-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 α2-AR subtypes have been defined. The α2A-AR and α2B-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, α2C-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 α2D-AR, has been identified in the rat and cow (20,21). This pharmacological subtype is a species ortholog of the human α2A-AR subtype and, thus, is not considered to be a separate genetic subtype. 1.1.3. β-AR Subtypes The β1-AR and β2-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 β1-AR and β2-AR have been subsequently developed. More recently, it became apparent that not all of the β-AR-mediated responses can be classified as either β1-AR or β2-AR, and thus, the β3-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 α2-AR α1-AR β1-AR β2-AR β3-AR β4-AR α1A-AR α1B-AR α1D-AR α2A-AR α2B-AR α2C-AR Library Construction 5 5 Table 1 α1-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence α1A-AR Oryctolagus cuniculus α1a-adrenoceptor U81982 cDNA (c) No 1401 bp rabbit liver (1..1401) O. cuniculus α1c-adrenoceptor subtype S75999 cDNA (p) No 432 bp rabbit brain (1..430) Homo sapiens adrenergic α1c receptor U03866 cDNA (c) No 1500 bp human prostate (66..1466) H. sapiens α1C-AR L31774 cDNA (c) No 1401 bp human prostate (1..1401) H. sapiens α1C-AR D25235 cDNA (c) No 2290 bp human prostate (437..1837) H. sapiens α1C-AR U02569 cDNA (c) No 1902 bp human prostate (425..1825) H. sapiens α1A-AR AF013261 cDNA (c) No 1765 bp human isoform 4 prostate (201..1568) H. sapiens α1C-adrenoceptor receptor D32201 cDNA (c) No 2089 bp human isoform 3 prostate (437..1726) H. sapiens α1C-AR D32202 cDNA (c) No 2306 bp human isoform 2 prostate (437..1936) H. sapiens α1c-adrenoceptor subtype S76001 cDNA (p) No 432 bp human brain, (1..431) saphenous vein (continued) 6 Scofield, Deupree, and Bylund Table 1 (continued) α1-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence H. sapiens α1a-AR U72653 Genomic Yes 6195 bp human (no coding region) Bos taurus α1C-AR J05426 cDNA (c) No 2461 bp cow adult brain (97..1497) cortex Mus musculus α1a-AR S80220 cDNA (p) No 252 bp house mouse brain (1..252) M. musculus α1A-AR AF031431 cDNA (c) No 1401 bp house mouse brain, liver, (1..1401) kidney Rattus norvegicus α1C-AR U13368 cDNA (c) No 1862 bp Norway rat cardiac (36..1436) myocytes R. norvegicus α1c-AR U07126 cDNA (c) No 1428 bp Norway rat brain (16..1416) Meriones unguiculatus α1a-AR AF047188 cDNA (p) No 175 bp Mongolian gerbil spiral modiolar (1..175) artery Oryzias latipes α1A adrenoceptor D63859 Genomic (c) No 1770 bp Japanese medaka fish (228..1640) α1B-AR M. musculus α1B-AR Y12738 cDNA (c) No 2268 bp house mouse brain (724..2268) 6 Library Construction 7 Mus. musculus α1b-AR S80219 cDNA (p) No 120 bp house mouse brain (3..19) R. norvegicus α1B-AR X51585 cDNA (c) No 2086 bp Norway rat brain (241..1788) R. norvegicus α1B-AR M60655 cDNA (c) No 2108 bp Norway rat brain (15..1562) R. norvegicus α1B-AR D32045 Genomic Yes 2387 bp Norway rat (1628..2387) R. norvegicus α1B-AR U83985 Genomic Yes 513 bp Norway rat (no coding region) R. norvegicus α1B-AR L08609 Genomic (p) Yes 2207 bp Norway rat exon 1 (1209..2157, L08610:51..649) R. norvegicus α1B-AR L08610 Genomic (p) No 1365 bp Norway rat exon 2 (L08609: 1209..2157, 51..649) M. unguiculatus α1b-AR AF047189 cDNA (p) No 258 bp Mongolian gerbil spiral modiolar artery (1..258) Mesocricetus auratus α1B-AR J04084 cDNA (c) No 2089 bp Syrian golden hamster smooth muscle (15..1562) H. sapiens adrenergic α1b receptor U03865 cDNA (c) No 1738 bp human brainstem (124..1686) H. sapiens α1B-AR L31773 cDNA (c) No 1560 bp human heart (1..1560) H. sapiens α1B-AR M99589 Genomic Yes 2669 bp human (no coding region) (continued) 7 8 Scofield, Deupree, and Bylund Table 1 (continued) α1-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence Canis familiaris RDC5 mRNA for G protein- X14050 cDNA (p) No 1695 bp dog coupled receptor thyroid (1..1256) α1D-AR R. norvegicus α1A-AR M60654 cDNA (c) No 2936 bp Norway rat brain (480..2162) R. norvegicus α1a/d-AR L31771 cDNA (c) No 2939 bp Norway rat cerebral cortex (480..2165) R. norvegicus α1D-AR AF071014 Genomic (p) Yes 1783 bp Norway rat (1597..1783) M. musculus α1d-AR S80044 cDNA (c) No 1902 bp house mouse brain (118..1806) M. musculus α1A-AR L20333 cDNA (p) No 483 bp house mouse homolog testis (1..483) H. sapiens α1a/d-AR L31772 Genomic and No 1831 bp human cDNA (c) (1..1719) prostate H. sapiens adrenergic α1a receptor U03864 cDNA (c) No 1860 bp human hippocampus (58..1776) H. sapiens α1A/D-AR D29952 Genomic and No 2077 bp human cDNA (c) (5..1723) prostate H. sapiens αA1-AR M76446 cDNA (c) No 2002 bp human brain (56..1561) O. cuniculus α1d adrenoceptor U64032 cDNA (c) No 1731 bp rabbit liver (1..1731) aAssignment of subtype is based on the alignment and groupings of coding sequences generated by the denogram of the program Pileup, GCG. b(c) Complete coding sequence; (p) partial coding sequence. 8 Library Construction 9 Table 2 α2-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence α2A-AR Rattus rattus α2D-AR U79031 cDNA (c) No 1552 bp black rat brain (1..1353) R. norvegicus α2-AR-RG20 M62372 Genomic (c) No 1380 bp Norway rat (1..1353) R. norvegicus α2D-AR U49747 Genomic (p) Yes 2836 bp Norway rat (2831..2836) M. musculus α2-AR M99377 Genomic (c) No 1454 bp house mouse (α2-AR-C10 homolog) (51..1403) M. musculus α2A-AR U29693 Genomic Yes 2828 bp house mouse (no coding region) M. auratus α2 receptor adrenergic L28124 cDNA (p) No 313 bp golden hamster (α2A-AR) adipocytes (1..313) Sus scrofa α2A-AR J05652 Genomic (c) No 1728 bp pig (α2-AR-C10 homolog) (130..1482) Bos taurus α2D-AR U79030 Genomic (c) Yes 2923 bp cow (1509..2867) B. taurus adrenergic receptor S66295 cDNA (p) No 120 bp cow subtype α2D-AR pineal gland (1..120) H. sapiens α2-AR M18415 Genomic (c) No 1521 bp human (59..1411) H. sapiens α2-AR M23533 Genomic (c) Yes 3604 bp human (α-2A) (2078..3430) Gallus gallus adrenergic receptor S66185 Genomic (p) No 117 bp chicken subtype α2A-AR (1..117) (continued) 9 10 Scofield, Deupree, and Bylund Table 2 (continued) α2-Adrenergic Receptors Adrenergic Definition in GenBank cDNA or 5′ Number of receptor Species, database, subtype Accession genomicb Flanking nucleotides, subtypea common name identification in text number tissue type region coding sequence Cavia porcellus α2A-AR adrenoceptor U25722 Genomic (c) No 2291 bp guinea pig (49..1401) α2B-AR R. norvegicus α2B-AR M32061 cDNA (c) No 2319 bp Norway rat (RNG-α2-AR) kidney (366..1727) R. norvegicus α2B-AR X74400 Genomic (c) No 1639 bp Norway rat (178..1524) M. musculus α2-AR L00979 Genomic (c) No 1650 bp house mouse (α2C2-AR homolog) (227..1573) M. musculus α2-AR M94583 Genomic (c) Yes 5265 bp house mouse (α2C2-AR homolog) (1146..2513) Elephas maximus α2B-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 α2B-AR Y12523 Genomic (p) No 1168 bp cape rock hyrax (1..1168) (shrewmouse) Orycteropus afer α2B-AR Y12522 Genomic (p) No 1165 bp aardvark (1..1165) Amblysomus hottentotus α2B-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 α2B-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 α2B-AR Y12521 Genomic (p) No 1174 bp western European (1..1174) hedgehog Talpa europaea α2B-AR Y12520 Genomic (p) No 1192 bp European mole (1..1192) H. sapiens α2B-AR AF005900 Genomic (c) Yes 9842 bp human (α2C2-AR) (5398..6750) H. sapiens α2-AR M34041 Genomic (c) No 2072 bp human (α2-AR c2) (413..1765) H. sapiens α2-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 α2B 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) α2C-AR M. musculus α2-AR M97516 Genomic (c) No 2409 bp house mouse (α2-C4 homolog) (415..1791) M. musculus α2-AR M99376 Genomic (c) No 1503 bp house mouse (α2-C4 homolog) (51..1427) R. norvegicus α2-AR-RG10 M62371 Genomic (c) No 1380 bp Norway rat (1..1377) R. norvegicus α2-C4-AR X57659 Genomic and ? 2991 bp Norway rat cDNA (c) (907..2283) brain (continued) 11

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