Introduction:  Protein-protein and protein-lipid interactions in membranes play a critical role in regulating transmembrane signal transduction. Receptors, channels and other integral membrane proteins reside in equilibrium between resting and active, ligand-bound state, the latter of which is responsible for activating distinct intracellular signaling pathways. A key mechanism by which this equilibrium is regulated is through transmembrane-mediated receptor oligomerization. We are interested in studying the regulated oligomerization of a class of membrane co-receptors, receptor activity modifying proteins (RAMPs), with the G-protein coupled receptor calcitonin-like receptor (CLR) during early heart development, focusing on the role of transmembrane domain interactions in regulating signaling. RAMP1-CLR plays numerous roles in heart development and vascular maintenance in adults, and dysregulation of the RAMP1-CLR signaling has been linked to numerous disease states, including acute migraines and acute coronary syndrome (ACS), and small-molecule therapeutics targeting CLR-RAMP heterooligomers are currently in clinical trials as a treatment for migraines. Thus, understanding the structural basis for CLR-RAMP heterooligomerization is central to understanding the basis for CLR signaling in vascular development as well as in developing effective approaches to treating diseases associated with CLR.Results and Discussion:  Using sequence-directed searches of transmembrane structural databases, we identified multiple interfaces common to all co-receptors and CLR and developed a structural model linking these interfaces to the ability to heterooligomerize with CLR. To validate this model, we developed a series novel, cell-based assays (Su and Berger, JBC 2012; Su and Berger, JMB 2013) that enable characterization of homo- and heterodimeric interfaces for single transmembrane helices in cell membranes, and used these assays for RAMP1-CLR. Biochemically, we confirmed the importance of the proposed heterodimeric interface in transfected cells by monitoring cAMP expression levels and in vivoFRET measurements, all of which demonstrated that targeted mutations to the predicted interface inhibited oligomerization of RAMP-CLR and attenuated cAMP expression. MO-mediated knockdown and RAMP1 rescue studies using zebrafish also confirm the importance of RAMP1 as well as the key role of the predicted interface in regulating multiple, distinct signaling events during cardiovascular development.Conclusions:  Overall, our results point to a central role for transmembrane-mediated interactions in regulating RAMP1-CLR heterooligomerization as well as a specific interface responsible for regulating signaling. Our current work is focused on implementing a robust, large-scale expression system (Su et al., Protein Science 2013) that will allow us to investigate in detail the structural basis for RAMP1-CLR heterooligomerization as well as design and characterization of peptide mimics that target the RAMP1-CLR interface.Acknowledgements:  This work was supported by the National Science Foundation (CBET-1227924) as well as the Anderson Medical Endowment.

Sequence, structure, and specificity in transmembrane signal transduction

247th National Meeting (2014)

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