Intracellularly truncated human alpha2B-adrenoceptors: stable and functional GPCRs for structural studies.
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.
Jaakola
Veli-Pekka
VP
Institute of Biotechnology, Biocenter 3, University of Helsinki, Helsinki, Finland.
Vainio
Minna
M
Sen
Saurabh
S
Rehn
Maria
M
Heimo
Heikki
H
Scheinin
Mika
M
Goldman
Adrian
A
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
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