Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

Abstract

Two cDNA clones, X RHM-MF and X RHB-DAR, encoding the muscarinic cholinergic receptor and the 13-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart P-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac j8 receptor has 88.0% homology (92.5% with favored substitutions) with the human brain f3 receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and fi-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.Pharmacological and physiological classification of receptors has grouped muscarinic and nicotinic cholinergic receptors together in a class distinctly separate from the adrenergic receptors. Our studies over the past several years have yielded immunological and biochemical data suggesting substantial structural similarity among 81-, /82-, a1-and a2-adrenergic receptors and muscarinic cholinergic receptors (1-3). The extent of structural homology among these proteins became more apparent with the cloning and sequence analysis of genes encoding ,B-adrenergic receptors from human brain (4), hamster lung (5), and turkey erythrocytes (6) and muscarinic cholinergic receptors from porcine heart (7, 8) and brain (9) (for review see ref. 10).In a comprehensive review (11) concerning the evolution of adrenergic and cholinergic receptors, we postulated that the muscarinic cholinergic receptor has existed for >600 million years. Pharmacological and biochemical evidence suggests that the B-adrenergic receptor may have appeared later in evolution raising the possibility that it evolved as a result of gene duplication.We have cloned and determined the primary structure of the genes encoding ,B-adrenergic and muscarinic cholinergic receptors from rat heart.t To our knowledge, this is the first report on the cloning of a cardiac /3-adrenergic receptor, and these sequences have allowed the first direct sequence comparison between an adrenergic and a muscarinic cholinergic receptor from the same species and tissue. For these studies we utilized automated DNA sequence analysis (12, 13), a technique that will hav...

Location of a region of the muscarinic acetylcholine receptor involved in selective effector coupling

“…In contrast, the exchange of this region between mAChR I and mAChR II does not significantly;affect the antagonist binding proper-ties of the two mAChR subtypes. The amino acid sequence of the putative cytoplasmic portion between the proposed transmembrane segments V and VI is divergent among the mAChR subtypes [2,12,[16][17][18]30,31].…”

The Sequence of the Human Genome

“…In 1986, Hood and co-workers (5) described an improvement in the Sanger sequencing method that included attaching fluorescent dyes to the nucleotides, which permitted them to be sequentially read by a computer. The first automated DNA sequencer, developed by Applied Biosystems in California in 1987, was shown to be successful when the sequences of two genes were obtained with this new technology (6). From early sequencing of human genomic regions (7), it became clear that cDNA sequences (which are reverse-transcribed from RNA) would be essential to annotate and validate gene predictions in the human genome.…”

Mutagenesis of the beta2-Adrenergic Receptor: How Structure Elucidates Function

“…Mutants constructed with deletions of amino acids 179-187 in the second extracellular loop bound agonist and antagonist with reduced affinity, perhaps implying a role of this region either in the direct binding of the ligand or in the proper folding and transmembrane assembly of the receptor (4,25). Later studies demonstrated that four cysteine residues (Cys 1 06, Cys 1 84, Cys190, and Cys191) located in the extracellular domains of the f3zAR are involved in disulfide-bridging, important for agonist and antagonist interactions with the receptor (25)(26)(27).…”

“…The cloning of .B2AR cDNA from hamster (2), human (3), rat (4,5), and mouse (6), as well as the gene from human genomic DNA (7,8), has allowed deduction of the amino acid sequence and analysis of the primary structure of these proteins. The amino acid sequence and proposed membrane topography for the human f32AR is presented in Figure 1.…”