Fellowship: Garcia-Cazarin: Study of the Trafficking and Regulatory Properties of the Alpha1D-Adrenoceptor

The alpha 1-adrenoceptors (alpha 1-ARs) regulate vascular tone and cardiac function. Abnormalities with the alpha1-AR contribute to the pathophysiology of hypertension and heart failure. Of the three alpha1-ARs, the alpha1 D-AR is the least studied. The alpha1D-AR is localized intracellularly and is refractory to agonist mediated internalization or desensitization. Considering the role of the alpha1D-AR in blood pressure regulation and hypertension a better understanding of its regulatory activities is critical. In Specific Aim #1, I hypothesize that the unique localization and trafficking properties of the alpha 1D-AR occur because it lacks crucial carboxyl-terminal sequences controlling these processes. This hypothesis will be tested by making chimeric receptors between the alpha1B and alpha1D-AR. I expect that an alpha1D-AR with an alpha1B-AR carboxyl-terminus will have typical GPCR properties of cell membrane localization and agonist mediated internalization and desensitization when transfected into HEK 293 cells. In Specific Aim #2, I will generate truncations at various positions of the alpha 1D-AR carboxyl-terminus to determine what sequences are responsible for its unique trafficking properties. It is well known that receptor overexpression in heterologous systems does not always yield an accurate assessment of the regulatory properties of a given receptor. Therefore, it is important to perform these experiments in a more physiologically relevant cell line such as vascular smooth muscle cells. In Specific Aim #3, adenoviruses that express the alpha1-ARs will be used to confirm that the localization seen in heterologous expression systems also occurs in human aortic smooth muscle cells. Subsequently, I will use vascular tissue to determine if the unique cellular localization of the alpha1 D-AR is responsible for its specific coupling to vascular smooth muscle contraction. This proposal combines molecular and physiological studies that will lead to understand how the regulatory activities of the alpha 1D-AR contribute to the regulation of systemic arterial blood pressu