Solution-phase library screening for the identification of rare clones: Isolation of an α(1D)-adrenergic receptor cDNA

D. M. Perez, M. T. Piascik, R. M. Graham

Research output: Contribution to journalArticlepeer-review

333 Scopus citations

Abstract

α1-Adrenergic receptor (α1-AR) subtypes (α(1A) and α(1B)) play a critical role in vascular smooth muscle contraction and circulatory homeostasis. Transcripts for these guanine nucleotide-binding protein-coupled receptors are extremely low in abundance, however, and isolation of their cDNAs is difficult. We have developed a novel technique for identifying rare clones in a cDNA library, which has been used successfully to isolate a cDNA clone encoding an α(1D)-AR. A 564-bp polymerase chain reaction product encoding a region between the third and sixth transmembrane domains of the α(1D)-AR was first generated using rat brain mRNA as template and highly degenerate primers. The primers corresponded to those domains but contained mismatches to the α(1B)-AR sequences. A 3-kb transcript was identified with this polymerase chain reaction probe, by Northern analysis of rat hippocampus. However, traditional plaque hybridization failed to identify a cDNA in a rat hippocampus λgt10 library. By solution-phase screening of virtually the entire library, a cDNA containing a 3-kb insert was identified, amplified, and purified. This insert encodes a 560-amino acid protein corresponding to the topology of guanine nucleotide-binding protein-coupled receptors. This receptor has approximately 71% amino acid identity, in the transmembrane regions, to the hamster and rat α(1B)-ARs. Characterization of the receptor expressed in COS-7 cells, by ligand binding and photoaffinity labeling, revealed some of the characteristics of an α(1A)-AR. However, unlike α(1A)-ARs characterized previously in membrane preparations or in solubilized partially purified preparations, the expressed receptor could be extensively inactivated by chlorethylclonidine. In addition, it displays ligand-binding properties that are not consistent with an α(1A)-AR. This indicates that the cDNA clone that we have isolated encodes a novel α1-AR subtype, which we classify as the α(1D)-AR.

Original languageEnglish
Pages (from-to)876-883
Number of pages8
JournalMolecular Pharmacology
Volume40
Issue number6
StatePublished - 1991

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Fingerprint

Dive into the research topics of 'Solution-phase library screening for the identification of rare clones: Isolation of an α(1D)-adrenergic receptor cDNA'. Together they form a unique fingerprint.

Cite this