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Intracellularly truncated human alpha(2B)-adrenoceptors: Stable and functional GPCRs for structural studies
Tekijät: Jaakola VP, Vainio M, Sen S, Rehn M, Heimo H, Scheinin M, Goldman A
Kustantaja: TAYLOR & FRANCIS INC
Julkaisuvuosi: 2005
Lehti:: Journal of Receptors and Signal Transduction
Tietokannassa oleva lehden nimi: JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
Lehden akronyymi: J RECEPT SIG TRANSD
Vuosikerta: 25
Numero: 2
Aloitussivu: 99
Lopetussivu: 124
Sivujen määrä: 26
ISSN: 1079-9893
DOI: https://doi.org/10.1081/RRS-200068745
Tiivistelmä
All three alpha(2)-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 alpha(2B)-adrenoceptor (alpha(2B)-AR) was investigated with a series of 3i truncated constructs (Delta 3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated alpha(2B)-AR Delta 3i with various agonists and measured [S-35]GTP gamma S 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 [S-35]GTP gamma S assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for G(i)/G(o) protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some alpha(2B)-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 alpha(2B)-AR.
All three alpha(2)-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 alpha(2B)-adrenoceptor (alpha(2B)-AR) was investigated with a series of 3i truncated constructs (Delta 3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated alpha(2B)-AR Delta 3i with various agonists and measured [S-35]GTP gamma S 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 [S-35]GTP gamma S assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for G(i)/G(o) protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some alpha(2B)-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 alpha(2B)-AR.
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