MODULATION OF MONOCARBOXYLIC ACID TRANSPORTER KINETIC FUNCTION FOR STROKE THERAPY -
DESCRIPTION (provided by applicant): The over-arching goal of the proposed work is to characterize the precise molecular mechanism by which Protein Kinase A (PKA) regulates the function of Monocarboxylic Acid Transporter 1 (MCT1) in rat brain endothelial cells. We hypothesize that PKA regulates MCT1 through a multi-protein regulatory complex called a transducisome. This work is significant because cerebrovascular endothelial cell MCT1 is the sole facilitator of lactic acid transport across the blood brain barrier's cerebrovascular endothelium. Therefore it is important in brain metabolism where lactate is an important energy substrate, and during pathological conditions such as ischemic stroke, hyperglycemic stroke, and brain injury, where brain lactic acidosis has been shown to be a key indicator in the etiology of cell damage. The development of new pharmacological treatments for stroke and brain injury could be advanced by better understanding factors that control lactic acidosis. The organization of multi-protein signaling complexes, or transducisomes, is ubiquitous in coordinating the functions of PKA in numerous cell systems, but is not well characterized in the cerebrovascular endothelium; therefore, an important additional item of significance is to elucidate the mechanism by which a transducisome organizes PKA dependent MCT1 regulation in cerebrovascular endothelial cells. Our broad hypothesis is that MCT1 is part of a transducisome complex that coordinates its PKA dependent regulation by facilitating MCT1 phosphorylation and vesicular trafficking. This regulatory action is dependent upon dynamic changes in the Actin cytoskeleton which facilitates trafficking and serves as an anchor for the transducisome. In the hypothesized complex, the C-terminus of MCT1 binds to a PDZ adapter protein which is associated with an AKAP protein. The AKAP protein binds to a beta-adrenergic receptor, PKA, and links the transducisome to the morphologically dynamic Actin cytoskeleton. We have shown that Actin cytoskeletal morphing upon PKA activation is a required factor in the regulation of MCT1. The complex is proposed to function by facilitating the spatial organization of protein interactions required for MCT1 phosphorylation by PKA, and also by mediating PKA dependent vesicular trafficking of MCT1 in a pathway dependent on the PDZ adapter protein, and/or the AKAP, and cytoskeletal morphology. We have discovered that PKA stimulation by cAMP analogues elicits a bimodal effect on MCT1 function; inhibiting it at 3 hours post-trypsinization, and increasing it when assessed at 24 hours post-trypsinization. We hypothesize that this bimodal regulatory effect of PKA is caused by the time-dependent formation of the transducisome complex following cytoplasmic disruption by trypsin. By examining the effects of our independent variables at both 3 and 24 hour time points we will capitalize on this dichotomy to further elucidate details about the transducisome. The proposed experimentation for our first specific hypothesis will determine whether the MCT1 C or N-terminus interacts with a PDZ adapter or AKAP binding partner as a functional requirement for its regulation by PKA. The binding partner will be identified. Our second hypothesis will more closely examine the transducisome and its mechanism of function by showing whether PKA stimulation leads to MCT1 and transducisome protein phosphorylation, vesicular trafficking of MCT1, cytoskeleton-morphology dependent changes in MCT1 regulation, and cytoskeleton morphology dependent changes key protein-protein interactions within the transducisome. Finally, this work will be done in a student-centered environment at Colorado State University in Pueblo. Undergraduate and graduate students will make significant contributions to this work, both in the research laboratory, and in the classroom where there will be significant spill-over of the proposed research. Colorado State University-Pueblo is the only federally recognized Hispanic serving university in Colorado, and the proposed work will have a major impact in building capacity for minority student access to medical research in the region. This grant would significantly strengthen the research abilities of our institution and would expose a large group of diverse students to biometrically relevant and important research.
PUBLIC HEALTH RELEVANCE: The results of these studies will produce basic scientific information needed to better understand the mechanism by which Protein Kinase A (PKA) regulates Monocarboxylic Acid Transporter 1 (MCT1) in cerebrovascular endothelial cells. Understanding the regulation of MCT1 in these cells is fundamental for understanding the control of monocarboxylate levels in brain. This, in turn, is important for brain energy metabolism and for diseases such as stroke and brain injury where lactic acidosis is a key indicator of the severity of brain damage. Such knowledge will help us to begin developing concepts for inventing new pharmacological-based therapies for stroke and brain injury patients. The rationale for the research is that during stroke, brain cell damage resulting directly from lactic acidosis could be reduced by agents that prevent or reduce it. Our aims focus on understanding the regulation of cerebrovascular MCT1 because it is the major molecular determinant of cerebral lactic acid efflux from brain in stroke (Smith and Drewes, 2006). The research will build a foundation for extending and developing these new therapeutic approaches, and will make a significant contribution in the training of new biomedical research scientists from under-represented minority groups.
