The application will deal with the mechanisms and structural functional relationships in the interaction of arrestin with G-protein coupled receptors and clathrin. There are three Specific Aims. The first is to characterize the specificity and role of arrestins in receptor desensitization. In this Specific Aim the investigator will initially generate a number of important tools including arrestin specific antibodies, a series of cells either expressing low levels of arrestin or high levels of arrestin for studies of expression of arrestin and receptors and their interaction, dominant negative and dominant active mutants of arrestin, antisense constructs as well as antisense oligonucleotides and knock-out cells which lack the expression of a specific arrestin. These tools will be used to study several new receptors not previously analyzed by the applicant including the alpha1-B adrenergic receptor interaction with Gq/G11 using methods well established in the applicant's laboratory. The applicant will also extend his studies to the beta2 adrenergic receptor and the M2 muscarinic acetylcholine receptor. Expression, purification, and characterization of these receptors and their interaction with arrestin will be carried out by previously described methods. A new proposal is presented to study these receptors in membrane preparations, which would allow study without the necessity of generating large amounts of purified proteins.
Specific Aim 2 will determine the role of arrestin in receptor trafficking. In this aim the applicant will study the in vitro interaction of arrestins and clathrin. He will use receptor domains which presumably alter the figuration of arrestin and permit its interaction with clathrin. Experiments will also be carried out to determine whether clathrin is capable of regulating the association of arrestin with the receptor. The applicant proposes to study the time-course of release of arrestin from the receptor and coated pits and the relationship to the time-course of dephosphorylation of the receptor. The relationship between the development of clathrin association of arrestin with clathrin coated pits to receptor degradation will also be studied. Finally the relationship between arrestin and possible lysosomal degradation (during down-regulation) of the receptor will be studied. In the third Specific Aim the applicant will carry out structure function analyses of arrestin by methods previously established in his laboratory. These will include maltose fusions from arrestin domains and their interaction with mutant receptors expressed as GST fusion proteins. Similar kinds of analyses of mutants will be carried out using clathrin binding domains and maltose fusions of arrestin domains. In this specific aim the applicant will generate dominant active and dominant maltose of arrestin.
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