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.
Subject(s)
Cell Membrane/metabolism , GTP-Binding Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Receptors, Adrenergic, alpha-2/chemistry , Receptors, Adrenergic, alpha-2/metabolism , Adrenergic alpha-Antagonists/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , CHO Cells , Cricetinae , Humans , Ligands , Molecular Sequence Data , Plasmids , Receptors, Adrenergic, alpha-2/genetics , Sequence DeletionABSTRACT
The aminergic alpha(2b)-adrenergic receptor (alpha(2b)-AR) third intracellular loop (alpha(2b)-AR 3i) mediates receptor subcellular compartmentalization and signal transduction processes via ligand-dependent interaction with G(i)- and G(o)- proteins. To understand the structural origins of these processes we engineered several lengths of alpha(2b)-AR 3i into the third intracellular loop of the proton pump bacteriorhodopsin (bR) and produced the fusion proteins in quantities suitable for physical studies. The fusion proteins were expressed in the Archaeon Halobacterium salinarum and purified. A highly expressed fusion protein was crystallized from bicelles and diffracted to low resolution on an in-house diffractometer. The bR-alpha(2b)-AR 3i(203-292) protein possessed a photocycle slightly perturbed from that of the wild-type bR. The first half of the fusion protein photocycle, correlated with proton release, is accelerated by a factor of 3, whereas the second half, correlated with proton uptake, is slightly slower than wild-type bR. In addition, there is a large decrease in the pK(a), (from 9.6 to 8.3) of the terminal proton release group in the unphotolyzed state of bR-alpha(2b)-AR 3i as deduced from the pH-dependence of the M-formation. Perturbation of a cytoplasmic loop has thus resulted in the perturbation of proton release at the extracellular surface. The current work indicates that long-range and highly coupled intramolecular interactions exist that are capable of "transducing" structural perturbations (e.g., signals) across the cellular membrane. This gene fusion approach may have general applicability for physical studies of G-protein-coupled receptor domains in the context of the bR structural scaffold.