Our broad, long-term objective is to understand the structural basis of the functions of receptors for angiotensin II (Ang II). The type 1 (AT1) receptor mediates diverse intracellular responses to Ang II in the regulation of blood pressure, hydromineral balance, cardiac hypertrophy and cell proliferation. The AT1 receptor is a novel paradigm for study since it is a G protein-coupled receptor with the unique ability to directly activate, besides G proteins, tyrosine kinases. Our structure-function analysis has identified critical interactions of the AT1 receptor with the agonist Ang II and the competitive antagonist losartan. These studies have yielded valuable mutants with loss of specific function and ligand-independent activation. Interpretation of structural consequences of these mutations by using a molecular model provides important insight into the mechanism of activation, desensitization, internalization and differential tyrosine kinase activation by the AT1 receptor. A novel class of dominant-negative (dn) AT1 receptor mutants have indicated that oligomerization of AT1 receptor may be essential for some functions. A combination of site-directed mutagenesis, site-directed chemical modification, molecular pharmacology, cell signaling and molecular modeling studies will be used to investigate some novel aspects of AT1 receptor structure-function. ? ? The specific aims for this competing renewal are: (1) To validate the novel structural features predicted by the homology model of the AT1 receptor based on the crystal structure of rhodopsin; (2) To elucidate the structural basis of prolonged inhibition of the AT1 receptor by insurmountable antagonists which are therapeutically better than competitive antagonists; and (3) To dissect the molecular basis of dominant-negative AT1 receptor mutants.
Specific Aims 1 and 2 will use the experimental strategy employed in our previous studies. In addition, we will use site-directed cystein modification studies to obtain structural information. The dnmutants will be tested for non-productive G-protein interaction and oligomerization using co-immunoprecipitation and fluorescence resonance energy transfer studies. The dn-mutant effects on Ang Il-specific cellular function and proteome responses will be evaluated. Validation of the model is critical for identification of structural constraints regulating the functions of the AT1 receptor. A refined molecular model will allow future improvement of antagonists. A better understanding of the mechanism of insurmountable antagonism will provide insight into design of novel anti-hypertensive, cardio- and reno-protective drugs that harbor greater therapeutic benefits. The study of trans-dominant effects will lead to novel mechanisms of signal transduction through oligomerization. The dn-mutants may serve as important tools for discovering cross regulation of intracellular signal-transduction pathways that are relevant to patho-physiology.
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