All cells sense their environment through membrane proteins (e.g. receptors and ion channels) and regulate their physiological responses accordingly. Angiotensin receptors are involved in an array of physiological processes including fetal kidney development, electrolyte homeostasis, and blood pressure control. Angiotensin receptor activation has also been linked to cellular phenotypic changes and cell growth, gene expression of hormones, growth factors, cytokines, and activation of multiple intracellular signaling cascades. Numerous studies have revealed that the pathophysiology of certain cardiovascular and renal diseases in humans is a result of aberrant activation of angiotensin receptors. A detailed molecular understanding of how receptors perceive and transfer information across cellular membranes is lacking due to limited knowledge of membrane protein structures and forces regulating their function. A goal of our work is to develop a useful expression system for over-expressing channels, transporters, and receptors critical for renal homeostasis. This system will produce the amounts of protein required to determine the structure of receptors. Our approach is to convert a naturally occurring membrane protein expression system to general usage. Toward that goal we have engineered a series of prototype expression vectors that will drive expression of the angiotensin receptor gene by elements known to direct the high levels of transcription and translation of the bacterio-opsin gene in the Archaeon H. salinarum. We will also use this new expression system to express a portion of the angiotensin receptor as a fusion to BR and use this fusion protein as an antigen allowing production of angiotensin receptor antibodies. This work is feasible and represents new methodology that will be made generally available for the study of receptors, channels, and transporters.
| Jaakola, Veli-Pekka; Rehn, Maria; Moeller, Martina et al. (2005) G-protein-coupled receptor domain overexpression in Halobacterium salinarum: long-range transmembrane interactions in heptahelical membrane proteins. Proteins 60:412-23 |
