Phe310 in transmembrane VI of the alpha1B-adrenergic receptor is a key switch residue involved in activation and catecholamine ring aromatic bonding.
Pharmacophore mapping of adrenergic receptors indicates that the phenyl ring of catecholamine agonists is involved in receptor binding and activation. Here we evaluated Phe310, Phe311, and Phe303 in transmembrane VI (TMVI), as well as Tyr348 in TMVII of the alpha1B-adrenergic receptor (alpha1B-AR), which have been implicated in a catechol-ring interaction. Neither catecholamine docking studies nor mutagenesis studies of Phe311, Phe303, or Tyr348 supported a role for these residues in catechol-ring binding. By contrast, docking studies indicated that the Phe310 side chain is well positioned to interact with the catechol-ring, and substituted cysteine accessibility method studies revealed that the side chain of the 310, but not 311 residue, is both solvent accessible and directed into the agonist-binding pocket. Also, saturation mutagenesis of both Phe310 and Phe311 revealed for the former, but not for the latter, a direct relationship between side chain volume and agonist affinity, and that aromaticity is essential for wild-type agonist binding, and for both wild-type agonist potency and efficacy. Moreover, studies of Phe310 mutants combined with a previously described constitutively active alpha1B-AR mutant, A293E, indicated that although not required for spontaneous receptor isomerization from the basal state, R, to a partially activated conformation R', interaction of Phe310 with catecholamine agonists is essential for isomerization from R' to the fully activated state, R.
Chen
S
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Molecular Cardiology Unit, Victor Chang Cardiac Research Institute, St. Vincent's Hospital, Sydney 2010, Australia.
Xu
M
M
Lin
F
F
Lee
D
D
Riek
P
P
Graham
R M
RM
D015078:2-Hydroxyphenethylamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002395:Catecholamines / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006224:Cricetinae; D004837:Epinephrine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010627:Phenethylamines / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000502:physiology*; D017433:Protein Structure, Secondary; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism
C547716:ADRA1B protein, human; D002395:Catecholamines; D010627:Phenethylamines; D018340:Receptors, Adrenergic, alpha-1; C029261:phenethylamine; D014443:Tyrosine; D010649:Phenylalanine; C027140:N-methylphenylethanolamine; D015078:2-Hydroxyphenethylamine; D004837:Epinephrine