(53a) Regulating Calcitonin-Like Receptor Signaling Through Transmembrane Domain Interactions

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 (1, 3). 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 (2). 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 model linking these interfaces to the ability to heterooligomerize with CLR. Biochemically, we confirmed the importance of one interface in transfected cells using oligomerization assays, monitoring cAMP expression levels and in vivo FRET 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 during heart development as well as the key role of the predicted interface in regulating multiple, distinct signaling events during cardiovascular development. Our qPCR results from our human aortic valve samples and zebrafish studies both point to multiple signaling pathways affected downstream of RAMP1-CLR as well as potential differences in expression between control and disease samples.

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 in both human aortic valve samples and zebrafish embryos. Our current and future work is focused on developing a robust, large-scale expression system that will allow us to investigate in detail the structural basis for RAMP1-CLR heterooligomerization, which will give us greater insight into the molecular details of transmembrane-mediated CLR signaling.

References:

1. Moore D, Berger B, & DeGrado W (2008) Protein-protein interactions in the membrane: sequence, structural, and biological motifs. Structure 16(7):991-1001.

2. Brain SD & Grant AD (2004) Vascular actions of calcitonin gene-related peptide and adrenomedullin. Physiological Reviews 84(3):903-934.

3. Berger BW, et al. (2010) Consensus motif for integrin transmembrane helix association. Proceedings of the National Academy of Sciences 107(2):703-708.