A CLONING STRATEGY FOR G-PROTEIN-COUPLED HORMONE RECEPTORS - THE OVINE BETA(1)-ADRENERGIC RECEPTOR

Regulation of beta(1)-adrenergic receptors is unusual in developing an imals. For example, glucocorticoid-and thyroid hormone-responsiveness for several genes is seen in animals treated during fetal life but bet a(1)-responsiveness is not seen until after birth. In order to investi gate this at the transcriptional level, the ovine beta(1) receptor gen e was cloned from a sheep genomic library. An approach using high-stri ngency screening with cDNA probes and oligonucleotides from regions of human and rat genes conserved but unique to the beta(1) receptor but not to other seven transmembrane, G-protein-coupled receptors. Over 80 0000 clones were screened from which 40-50 positive clones were identi fied by each of the probes. There was, however, only a single clone wh ich was recognized by each of the probes. A 5-kb insert was subcloned and shown to contain sequences which hybridized to each of the probes. Using the restriction map of the rat beta(1) receptor, a 1.0-kb Pst1 internal fragment was further subcloned for sequence identification. C onfirmation of this fragment as the ovine beta(1) receptor was based o n homology of the beta(1) receptor from other species and tissue distr ibution of mRNA. Nucleotide sequence homology was 93% with the human b eta(1) receptor and 84% with rat. Amino acid sequence homology was >75 % and approached 100% in the transmembrane regions. The approach descr ibed represents a practical approach to cloning and identification of hormone receptors from the highly homologous members of the seven-tran smembrane, G-protein-coupled receptors.