title: An aspartate in the second extracellular loop of the α(1B) adrenoceptor regulates agonist binding.
abstract: The extracellular loops of the adrenoceptors present a potential therapeutic target in the design of highly selective adrenergic drugs. These regions are less conserved than the orthosteric binding site but have to date not been implicated in activation of adrenoceptors. A previously generated homology model identified an extracellular residue, D191, as a potential regulator of agonist binding. We have generated mutants of the α1B adrenoceptor replacing the charged aspartate, D191, as well as a potential interaction partner, K331, with uncharged alanines to observe effects on ligand binding and receptor activation. Significant 4-6 fold reductions in affinity for the endogenous agonists, epinephrine and norepinephrine were observed for receptors with the D191A mutation in the second extracellular loop. While changes in EC50 were observed, operational analysis yielded no apparent change in receptor activation. Based on these findings, we suggest that D191, in the second extracellular loop of the α1B adrenoceptor, acts as a 'point of first contact' for the receptor's endogenous agonists. Implication of the non-conserved extracellular regions of the receptor in agonist binding makes it a potential target for the design of highly selective drugs.
authors.affiliation: Department of Pharmacology, School of Medical Sciences, University of New South Wales, Kensington, NSW 2052, Australia.
authors.forename: Adrian P
authors.initials: AP
authors.lastname: Campbell
authors.affiliation: School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Iain J A
authors.initials: IJ
authors.lastname: MacDougall
authors.affiliation: Department of Pharmacology, School of Medical Sciences, University of New South Wales, Kensington, NSW 2052, Australia.
authors.forename: Renate
authors.initials: R
authors.lastname: Griffith
authors.affiliation: Department of Pharmacology, School of Medical Sciences, University of New South Wales, Kensington, NSW 2052, Australia. Electronic address: angela.finch@unsw.edu.au.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Finch
mesh_terms: D058646:Adrenergic alpha-1 Receptor Agonists / Q000494:pharmacology*; D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D001667:Binding, Competitive; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D058646:Adrenergic alpha-1 Receptor Agonists; D008024:Ligands; D018340:Receptors, Adrenergic, alpha-1; D001224:Aspartic Acid; D000409:Alanine
keywords: GPCR; Receptor activation; Second extracellular loop; α(1B) Adrenergic receptor