title: Domains for activation and inactivation in G protein-coupled receptors--a mutational analysis of constitutive activity of the adenosine A2B receptor.
abstract: G protein-coupled receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Most, if not all, GPCRs also display a basal level of biological response in the absence of such a stimulus. This level of so-called constitutive activity results from a delicate energy equilibrium that exists between the active and the inactive state of the receptor and is the first determinant in the GPCR activation mechanism. Here we describe new insights in specific regions of the adenosine A2B receptor that are essential in activation and inactivation. We developed a new screening method using the MMY24 S. Cerevisiae strain by which we were able to screen for constitutively inactive mutants receptors (CIMs). We applied this screening method on a mutagenic library of the adenosine A2B receptor, where random mutations were introduced in transmembrane domains four and five (TM4 and TM5) linked by extracellular loop 2 (EL2). The screen resulted in the identification of 22 single and double mutant receptors, all showing a decrease in constitutive activity as well as in agonist potency. By comparing these results with a previous screen of the same mutagenic library for constitutively active mutant receptors (CAMs), we discovered specific regions in this G protein-coupled receptor involved in either inactivation or activation or both. The results suggest the activation mechanism of GPCRs to be much less restricted to sites of high conservation or direct interaction with the ligand or G protein and illustrate how dynamic the activation process of GPCRs is.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Electronic address: m.c.peeters@lacdr.leidenuniv.nl.
authors.forename: Miriam C
authors.initials: MC
authors.lastname: Peeters
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Qilan
authors.initials: Q
authors.lastname: Li
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Rachel
authors.initials: R
authors.lastname: Elands
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Gerard J P
authors.initials: GJ
authors.lastname: van Westen
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Eelke B
authors.initials: EB
authors.lastname: Lenselink
authors.affiliation: Pharmaceutical Institute, Pharmaceutical Chemistry I, Pharmaceutical Sciences Bonn (PSB), University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
mesh_terms: D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary; D011897:Random Allocation; D043704:Receptor, Adenosine A2B / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D043704:Receptor, Adenosine A2B; D043562:Receptors, G-Protein-Coupled; D029701:Saccharomyces cerevisiae Proteins
keywords: Adenosine A(2B) receptor; Constitutively inactive mutant (CIM); G protein-coupled receptor; Receptor activation mechanism; Saccharomyces cerevisiae