The long-term objective of this proposal is to understand the cell and molecular biology of G-protein-linked receptors, through analysis of the adrenergic receptor family (alpha1, alpha2, beta1, beta2). We propose to investigate three, highly-integrated hypotheses central to G-protein-linked receptor biology and transmembrane signaling.
The specific aims span from the macromolecular organization and structure of G-protein-linked receptors to post-transcriptional and post-translational regulation of receptors. (1) The macromolecular and cellular localization of adrenergic receptors have functional significance. Indirect immunofluorescence (IIF) localization of beta2-adrenergic receptors in intact tissue culture cells reveals punctate staining patterns that suggest a macromolecular organization (i.e., """"""""clustering"""""""") of these transmembrane signaling elements. To investigate the nature of these clusters, we propose: (i) to develop new high-affinity specific antibodies directed against purified holoreceptors through high-level expression of receptor in transfected cells, receptor purification, and antibody production and additional domain-specific antibodies to peptides; (ii) to elucidate the composition of these clusters by a combination of immunocytochemistry and in situ chemical crosslinking analysis; and (iii) to study the dynamics of these receptor clusters during activation, desensitization and down-regulation. (2)Intramolecular disulfide groups play a critical role in the receptor and function. Beta- adrenergic receptors display thiol-dependent shifts in their Mr, reversible thiol-dependent activation, and conservation of cysteinyl residues. We propose to employ stably transfected L cells and baculovirus-infected Sf9 cells that express wild-type beta2-adrenergic receptors or receptor with defined point mutations in cys residues and newly synthesized radiolabeled, bifunctional thiol-specific probes to accomplish the following objectives: (i) establish the stoichiometry and identity of disulfide bridges and free sulfhydryl groups in wild-type beta2-adrenergic receptor; (ii) investigate the status of receptor cysteinyl residues in unstimulated and agonist-activated states; and (iii) probe via site-specific mutagenesis the role of individual cysteinyl residues in receptor expression,turnover, function, and structure. (3)Protein phosphorylation and mRNA destabilization are essential for agonist-induced desensitization and down- regulation. Beta2-adrenergic receptors are substrates for protein phosphorylation associated with agonist activation and their mRNA levels are sensitive to chronic stimulation by agonist. We propose to investigate receptor phosphorylation and mRNA stability to (i) define the temporal relationships and stoichiometry of agonist-induced desensitization and phosphorylation via metabolic labeling with [32p]pi and [35S]-methionine; (ii) investigate receptor expression and turnover in transfectant cells expressing receptors with specific mutations in canonical phosphorylation sites; and (iii) explore the mechanism(s) of agonist induced destabilization of receptor mRNA that accompanies down-regulation.
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