title: Cloning of the human alpha 1d-adrenergic receptor and inducible expression of three human subtypes in SK-N-MC cells.
abstract: We have cloned the human alpha 1d-adrenergic receptor (AR) and compared the pharmacological properties of the three recombinant human alpha 1-AR subtypes in SK-N-MC cells. SK-N-MC cells natively express a mixture of alpha 1-AR subtypes, and the use of an inducible expression system allowed us to directly compare the recombinant and native subtypes without concern for cell-specific processing or microenvironment. The human alpha 1d-AR was expressed from a cDNA/gene fusion construct cloned from human SK-N-MC cell cDNA and human genomic libraries. This receptor is deduced to contain 572 amino acids with 98% identity to the rat alpha 1d-AR in the transmembrane domains and, when expressed in human embryonic kidney 293 cells, has alpha 1-AR binding properties similar to those of the rat alpha 1d-AR. Norepinephrine increased inositol phosphate formation and mobilized intracellular Ca2+ in transfected 293 cells. Reverse transcription-polymerase chain reaction analysis of the three cloned human subtypes (alpha 1a, alpha 1b, and alpha 1d) in mRNA from SK-N-MC cells, which natively express alpha 1A- and alpha 1B-like pharmacology, showed abundant alpha 1a and alpha 1d but fewer alpha 1b transcripts. The three human clones were expressed in SK-N-MC cells using isopropyl-beta-D-thiogalactoside-inducible vectors. Upon induction, alpha 1-AR density was increased with the recombinant subtype comprising 67-80% of total alpha 1-ARs. Inhibition curves for (+)-niguldipine and 5-methylurapidil fit best to a two-site model in uninduced cells, indicating significant receptor heterogeneity. Isopropyl-beta-D-thiogalactoside induction altered the potencies of both compounds, causing most inhibition curves to fit best to a one-site model. (+)-Niguldipine was 100-fold more potent at the alpha 1a-AR than at alpha 1b- or alpha 1d-ARs, whereas 5-methylurapidil had similar potencies at alpha 1a- and alpha 1d-ARs and about 10-fold lower affinity at the alpha 1b-AR. We conclude that the complex alpha 1A- and alpha 1B-like pharmacology observed in native SK-N-MC cells is due to expression of all three subtypes in different proportions, independently of cell-specific processing or environmental factors, and that the alpha 1a-AR cDNA encodes the pharmacologically defined alpha 1A subtype.
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Esbenshade
authors.forename: A
authors.initials: A
authors.lastname: Hirasawa
authors.forename: G
authors.initials: G
authors.lastname: Tsujimoto
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
authors.forename: J
authors.initials: J
authors.lastname: Yano
authors.forename: K P
authors.initials: KP
authors.lastname: Minneman
authors.forename: T J
authors.initials: TJ
authors.lastname: Murphy
mesh_terms: D000317:Adrenergic alpha-Antagonists / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004095:Dihydropyridines / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010627:Phenethylamines / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D044024:Tetralones; D014162:Transfection
chemical_list: D000317:Adrenergic alpha-Antagonists; D017931:DNA Primers; D018076:DNA, Complementary; D004095:Dihydropyridines; D010627:Phenethylamines; D010879:Piperazines; D018340:Receptors, Adrenergic, alpha-1; D011994:Recombinant Proteins; D044024:Tetralones; C057446:5-methylurapidil; D007544:Isopropyl Thiogalactoside; C010652:BE 2254; C054074:niguldipine