Activation of beta-adrenergic receptors causes relaxation of bronchial smooth muscle and these receptors are the targets of the most effective therapy for asthma. Several lines of investigation suggest that defects in the beta-adrenergic receptor/G-protein/adenylyl cyclase system may be a contributory factor in asthma. The long term goals of this proposal are to determine the role of beta-adrenergic receptors in the pathogenesis of asthma, to elucidate the mechanisms and pathways of receptor regulation by various factors, and to establish relationships between receptor structure and function. First, the molecular structure of any additional beta- adrenergic receptor subtypes in lung will be determined by screening human lung cDNA libraries using probes from the known sequences of beta1-,beta2-, and beta3- adrenergic receptors and subsequent cloning. To assess whether a defect in the receptor structure is present in asthma, a large cohort of asthmatic patients will be studied. Genomic DNA will be isolated from blood samples and beta-adrenergic receptor sequences amplified by the polymerase chain reaction. The resulting receptor genes will be sequenced in their entirety. When matched with controls, this will allow for delineation of any unique abnormality in primary beta-adrenergic receptor structure present in asthmatics. to further explore the receptor- transduction system in human subjects, lung tissue from bronchoscopy, surgery or autopsy will be utilized. Receptor density on various pertinent cells (smooth muscle, endothelium, mucous glands, epithelium, macrophages) will be assessed with radioligand binding and autoradiography, and will be correlated with receptor mRNA levels using in situ hybridization and Northern blots. Under these conditions the expression of receptor at pertinent cells can be compared between normal and asthmatic lungs, and the relationship between expression and mRNA levels determined. It is also planned to establish permanent cell lines from explants of lungs from normal and asthmatic subjects which will allow for even further pharmacologic characterization of the receptor and various transduction pathways. To further define the pathways involved in regulation of beta- adrenergic receptor expression and function, it is planned to transfect the human beta-adrenergic gene into cells normally devoid of receptors, thus establishing permanent cell lines expressing the receptor. By site directed mutagenesis, receptors with altered regions thought to be involved in receptor function will be constructed and expressed. This will allow for a molecular assignment of determinants of several key regulatory events: homologous and heterologous desensitization, phosphorylation by PKA and the beta-adrenergic receptor kinase, agonist induced downregulation, receptor fatty acid acylation and G-protein coupling.
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