title: Intracellularly truncated human alpha2B-adrenoceptors: stable and functional GPCRs for structural studies.
abstract: All three alpha2-adrenoceptor subtypes have a long third intracellular loop (3i), which is conserved by overall size and charge-hydrophobic properties but not by amino acid sequence similarity. These properties must be relevant for function and structure, because they have been preserved during hundreds of millions of years of evolution. The contribution of different loop portions to agonist/antagonist binding properties and G protein coupling of the human alpha2B-adrenoceptor (alpha2B-AR) was investigated with a series of 3i truncated constructs (delta3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated alpha2B-AR delta3i with various agonists and measured [35S]GTPgammaS binding in isolated cell membranes with or without antagonist inhibition. We also evaluated the ability of oligopeptides, analogous to the amino and carboxyl terminal parts of 3i, to promote G protein activation, monitored with the [35S]GTPgammaS assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for Gi/Go protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some alpha2B-AR agonists, indicating that agonist binding is dependent on the conformation of the 3i domain, possibly through the involvement of G protein interactions. The truncated receptor constructs may be more stable on purification and thus be useful for structural characterization of alpha2B-AR.
authors.affiliation: Institute of Biotechnology, Biocenter 3, University of Helsinki, Helsinki, Finland.
authors.forename: Veli-Pekka
authors.initials: VP
authors.lastname: Jaakola
authors.forename: Minna
authors.initials: M
authors.lastname: Vainio
authors.forename: Saurabh
authors.initials: S
authors.lastname: Sen
authors.forename: Maria
authors.initials: M
authors.lastname: Rehn
authors.forename: Heikki
authors.initials: H
authors.lastname: Heimo
authors.forename: Mika
authors.initials: M
authors.lastname: Scheinin
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman
mesh_terms: D000317:Adrenergic alpha-Antagonists / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: C547771:ADRA2B protein, human; D000317:Adrenergic alpha-Antagonists; D008024:Ligands; D018341:Receptors, Adrenergic, alpha-2; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins