CLONING OF THE HUMAN ALPHA(1D)-ADRENERGIC RECEPTOR AND INDUCIBLE EXPRESSION OF 3 HUMAN SUBTYPES IN SK-N-MC CELLS

We have cloned the human alpha(1d)-adrenergic receptor (AR) and compar ed the pharmacological properties of the three recombinant human alpha (1)-AR subtypes in SK-N-MC cells. SK-N-MC cells natively express a mix ture of alpha(1)-AR subtypes, and the use of an inducible expression s ystem allowed us to directly compare the recombinant and native subtyp es without concern for cell-specific processing or microenvironment. T he 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 th e 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 ino sitol phosphate formation and mobilized intracellular Ca2+ in transfec ted 293 cells. Reverse transcription-polymerase chain reaction analysi s 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) b ut fewer alpha(1b) transcripts. The three human clones were expressed in SK-N-MC cells using isopropyl-beta-D-thiogalactoside-inducible vect ors. Upon induction, alpha(1)-AR density was increased, with the recom binant subtype comprising 67-80% of total alpha(1)-ARs. Inhibition cur ves for (+)-niguldipine and 5-methylurapidil fit best to a two-site mo del in uninduced cells, indicating significant receptor heterogeneity. Isopropyl-beta-D-thiogalactoside induction altered the potencies of b oth compounds, causing most inhibition curves to fit best to a one-sit e model. (+)-Niguldipine was 100-fold more potent at the alpha(1a)-AR than at alpha(1b)- or alpha(1d)-ARs, whereas 5-methylurapidil had simi lar 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 du e to expression of all three subtypes in different proportions, indepe ndently of cell-specific processing or environmental factors, and that the alpha(1a)-AR cDNA encodes the pharmacologically defined alpha(1A) subtype.