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TA Esbenshade, A Hirasawa, G Tsujimoto, T Tanaka, J Yano, KP Minneman and TJ Murphy
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
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.
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