title: The role of the second and third extracellular loops of the adenosine A1 receptor in activation and allosteric modulation.
abstract: The adenosine A1 receptor is a member of the large membrane protein family that signals through G proteins, the G protein-coupled receptors (GPCRs). GPCRs consist of seven transmembrane domains connected by three intracellular and three extracellular loops. Their N-terminus is extracellular, the C-terminal tail is in the cytoplasm. The transmembrane domains in receptor subfamilies that bind the same endogenous ligand, such as dopamine or adenosine, tend to be highly similar. In contrast, the loop regions can vary greatly, both in sequence and in length, and the role these loops have in the activation mechanism of the receptors remains unclear. Here, we investigated the activating role of the second and third extracellular loop of the human adenosine A1 receptor. By means of an (Ala)3 mutagenic scan in which consecutive sets of three amino acids were mutated into alanine residues in EL2 and a classical alanine scan in EL3, we revealed a strong regulatory role for the second extracellular loop (EL2) of the human adenosine A1 receptor. Besides many residues in the second and the third extracellular loops important for adenosine A1 receptor activation, we also identified two residues in EL2, a tryptophan and a glutamate, that affect the influence of the allosteric modulator PD81,723. These results, combined with a comparison of the different receptor loop regions, provide insight in the activation mechanism of this typical class A GPCR and further emphasize the unique pharmacological profile the loops can provide to individual receptors, even within subfamilies of GPCRs.
authors.affiliation: Division of Medicinal Chemistry, Leiden/Amsterdam Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: M C
authors.initials: MC
authors.lastname: Peeters
authors.forename: L E
authors.initials: LE
authors.lastname: Wisse
authors.forename: A
authors.initials: A
authors.lastname: Dinaj
authors.forename: B
authors.initials: B
authors.lastname: Vroling
authors.forename: G
authors.initials: G
authors.lastname: Vriend
authors.forename: A P
authors.initials: AP
authors.lastname: Ijzerman
mesh_terms: D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D043682:Receptor, Adenosine A1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D013876:Thiophenes / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: D008024:Ligands; D043682:Receptor, Adenosine A1; D013876:Thiophenes; C075004:PD 81723; D018698:Glutamic Acid; D014364:Tryptophan; D000409:Alanine