Beta-agonists remain a cornerstone in the treatment of asthma. Beta2- adrenergic receptors (beta2AR) are expressed on many cell types in the lung which are involved in the pathogenesis and/or treatment of asthma. Results using a number of approaches have demonstrated that beta2AR dysfunction can accompany asthma. This project is directed at further understanding the molecular basis of receptor function and regulation, and the mechanisms by which receptor dysfunction can accompany asthma. The focus will be on two general mechanisms that have been identified as important in modifying receptor function: agonist exposure (desensitization) and genetic variation in the structure of the beta2AR gene.
In Specific Aim 1, we will delineate which of the five known G-protein coupled receptor kinases (GRKs) phosphorylate the beta2AR during agonist exposure. Such phosphorylation plays an important role in both short- and long-term agonist-promoted desensitization of the receptor, and knowing which kinases subserve this function is critical to our understanding of how chronic beta-agonist therapy in asthma can lead to tachyphylaxis. For this aim, we have developed an approach using recombinant coexpression of beta2AR and individual GRKs which provides for a means to determine whether a given kinase phosphorylates and induces desensitization of the receptor.
For Specific Aim 2, the serine or threonine residues in the beta2AR which are phosphorylated by GRKs during agonist exposure will be determined by site-directed mutagenesis and recombinant expression. Results from this aim and from those of Specific Aim 1 will provide the molecular mechanism for agonist-promoted desensitization of the beta2AR and a basis for developing a strategy to block tachyphylaxis to beta- agonist treatment in asthma.
In Specific Aim 3, the distribution of the GRKs will be determined in human lung. Preliminary studies using in situ hybridization have demonstrated marked cell-type heterogeneity of the expression of these kinases. Delineating the distribution of these key kinases will further our understanding of how beta2AR are regulated on different cells in the lung by administration of beta-agonists. The critical features involved with agonist activation of the receptor will be further explored in Specific Aim 4, where the molecular requirements for agonist and antagonist binding of the beta2AR will be determined by site- directed mutagenesis, recombinant expression, and pharmacologic characterization. Polymorphisms of the 5' untranslated region and the coding block of the beta2AR gene have been recently identified, with some coding block polymorphisms resulting in altered beta2AR phenotypes.
In Specific Aim 5, the identity and biological relevance of polymorphisms in the 5' untranslated region of the beta2AR gene will be assessed in genomic DNA samples obtained from asthmatics and normal subjects. The functional significance of such polymorphisms in regard to gene transcription will be delineated using mutagenesis/reporter gene and biochemical techniques.
Specific Aim 6 involves studies to delineate the role of beta2AR gene polymorphisms in defining asthmatic phenotypes. This will be carried out in a case-control study of fatal asthma and in a classic genetic family study. The results of the this effort will help to define the role of beta2AR polymorphisms in determining the severity of the asthma, its response to beta-agonist therapy, and other phenotypic characteristics.
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