Molecular cloning and characterization of a novel beta-adrenergic receptor

In an attempt to isolate new G protein-coupled receptors from turkey erythrocytes, reverse transcribed polymerase chain reaction was performed on fetal turkey blood RNA using degenerate primers based on conserved sequences present in seven transmembrane receptors. An open reading frame in one of the clones, designated 4C (497 base pairs), displayed approximately 50-60% identity to all of the previously cloned beta-adrenergic receptors (beta-ARs). A lambda-DASH turkey genomic library was screened with a probe generated from the partial 4C cDNA, and the gene encoding this receptor was localized to a 3.5-kilobase pair HindIII fragment. Ribonuclease protection analysis of turkey lung mRNA indicated that the 3' end of the coding sequence of the 4C gene, like beta 3-AR, was interrupted by an intron. To obtain the cDNA sequence of 4C, RNA-polymerase chain reaction was performed using primers complementary to regions identified by ribonuclease protection analysis to be present in 4C mRNA. Comparison of the genomic and cDNA sequences of 4C indicated that the first exon encodes 414 amino acids of the protein, the second exon (68 base pairs) encodes an additional 12 residues followed by a stop codon, and the third exon is composed of 3'-untranslated sequence. The 4C receptor was transiently expressed in COS-1 cells, and the apparent affinities of a series of beta-AR agonists and antagonists were determined using [125I]iodocyanopindolol. As implicated by its amino acid sequence, 4C displayed a pharmacological selectivity that was consistent with that of a beta-AR but distinct from other cloned beta-ARs. Isoproterenol, epinephrine, and norepinephrine stimulated cyclic AMP accumulation in a concentration-dependent manner in mouse L cells stably expressing the 4C receptor. No effect on phospholipase C activity was observed. Ribonuclease protection assays indicated that 4C mRNA exhibits a broad tissue distribution, which suggests that it may play an important role in avian physiology.