Solution-phase library screening for the identification of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA.
alpha 1-Adrenergic receptor (alpha 1-AR) subtypes (alpha 1A and alpha 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 alpha 1D-AR. A 564-bp polymerase chain reaction product encoding a region between the third and sixth transmembrane domains of the alpha 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 alpha 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 lambda gt10 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 alpha 1B-ARs. Characterization of the receptor expressed in COS-7 cells, by ligand binding and photoaffinity labeling, revealed some of the characteristics of an alpha 1A-AR. However, unlike alpha 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 alpha 1A-AR. This indicates that the cDNA clone that we have isolated encodes a novel alpha 1-AR subtype, which we classify as the alpha 1D-AR.
Perez
D M
DM
Department of Heart and Hypertension Research, Cleveland Clinic Foundation, Ohio 44195-5071.
Piascik
M T
MT
Graham
R M
RM
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002417:Cattle; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015698:Genomic Library; D006624:Hippocampus / Q000737:chemistry / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011224:Prazosin / Q000378:metabolism; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000235:genetics
D003062:Codon; D011942:Receptors, Adrenergic, alpha; D004247:DNA; D011224:Prazosin