Abstract
Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.
Highlights
Previous studies have shownthat differences in sub- distinguish between p2adrenergic receptor antagonists and a2 type-specific ligand binding between a2 and & adre- adrenergic receptor antagonists are found predominantly in nergic receptors are largelydetermined by the seventh the seventh hydrophobic domain [6].This paper describes the hydrophobic domain
The mutant receptorsa,F"+N and 0,N-F were constructed by polymerase chain reaction using oligonucleotide primers with base mismatches [7]
Sequence Comparison-Fig. 1 shows a comparison of the amino acid sequences for the seventh hydrophobic domains of the a and P adrenergic receptors cloned to date
Summary
Previous studies have shownthat differences in sub- distinguish between p2adrenergic receptor antagonists and a2 type-specific ligand binding between a2 and & adre- adrenergic receptor antagonists are found predominantly in nergic receptors are largelydetermined by the seventh the seventh hydrophobic domain [6].This paper describes the hydrophobic domain. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of j3 receptor antagonists. Expression-Wild-type and mutant receptor DNA sequences were cloned into the expression vector pBC12MI [8] and were transiently expressed in COS-7 cells using a DEAE-dextran transfection procelieved to interact with the negatively charged carboxyl group dure [6].
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