title: Allosteric modulation and constitutive activity of fusion proteins between the adenosine A1 receptor and different 351Cys-mutated Gi alpha-subunits.
abstract: We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated G(i1) alpha-subunits (A1-Gialpha) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first analysed whether such fusion products are still subject to allosteric modulation, and, secondly, we investigated the potential utility of the fusion proteins to study constitutive receptor activity. We determined the pharmacological profile of nine different A1-Gialpha fusion proteins in radioligand binding studies. In addition, we performed [35S]GTPgammaS binding experiments to study receptor and G protein activation of selected A1-Gialpha fusion proteins. Compared to unfused adenosine A1 receptors, the affinity of N6-cyclopentyladenosine (CPA) at wild-type A1-Gialpha fusion proteins (351Cys) increased more than eightfold, while the affinity of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not change significantly. Furthermore, we showed that the allosteric enhancer of agonist binding, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)-phenyl]methanone), elicited similar effects on ligand binding; i.e. CPA binding to the A1-Gialpha fusion proteins was enhanced, whereas the affinity of DPCPX was hardly affected. Moreover, sodium ions were unable to decrease agonist binding to the majority of the A1-Gialpha fusion proteins, presumably because they exhibit their effect through uncoupling of the R-G complex. From [35S]GTPgammaS binding experiments, we learned that all the A1-Gialpha fusion proteins tested had a higher basal receptor activity than the unfused adenosine A1 receptor, thereby providing improved conditions to observe inverse agonism. Moreover, the maximal CPA-induced stimulation of basal [35S]GTPgammaS binding was increased for the five A1-Gialpha fusion proteins tested, whereas the inhibition induced by 8-cyclopentyltheophylline (CPT) was more pronounced at 351Cys, 351Ile, and 351Val A1-Gialpha fusion proteins. Thus, the maximal receptor (de)activation depended on the amino acid at position 351 of the Gi alpha-subunit. In conclusion, A1-Gialpha fusion proteins, especially with 351Cys and 351Ile, can be used as research tools to investigate inverse agonism, due to their increased readout window in [35S]GTPgammaS binding experiments.
authors.affiliation: Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Klaasse
authors.forename: Rianne A F
authors.initials: RA
authors.lastname: de Ligt
authors.forename: Sophie F
authors.initials: SF
authors.lastname: Roerink
authors.forename: Anna
authors.initials: A
authors.lastname: Lorenzen
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
authors.forename: Ad P
authors.initials: AP
authors.lastname: IJzerman
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000495:Allosteric Site / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011869:Radioligand Assay; D043682:Receptor, Adenosine A1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D013876:Thiophenes / Q000494:pharmacology; D014162:Transfection; D014316:Tritium; D014970:Xanthines / Q000378:metabolism
chemical_list: D043682:Receptor, Adenosine A1; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D013876:Thiophenes; D014970:Xanthines; D014316:Tritium; C075004:PD 81723; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C048599:N(6)-cyclopentyladenosine; D012965:Sodium Chloride; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000241:Adenosine; D003545:Cysteine