Monocyte migration into tissues, guided by inflammatory chemokine CCL2 and its G protein-coupled receptor CCR2, is key to both normal physiological responses and many diseases including neurodegenerative disease, traumatic brain injury, arthritis, diabetic nephropathy and non-alcoholic fatty liver disease. Moreover, CCR2 is currently the target of ongoing clinical trials for diabetic nephropathy and pancreatic cancer. Given the therapeutic significance of CCR2, it is surprising that its role in monocyte function is so poorly understood. In particular, while CCR2 is well-recognized to promote cell movement, what is largely unappreciated, is that it has a second, equally important function, whereby it scavenges chemokines from the extracellular medium by internalizing and trafficking chemokine to lysosomes for degradation. Scavenging may enable receptor- expressing cells to move along gradients of increasing chemokine concentration without desensitizing; it may also modulate the responsiveness of receptors on different cells by altering the availability of specific chemokines, thereby regulating whether and what type of cells migrate. We hypothesize that it is, in fact, the cohesive integration of these two functions (cell movement and scavenging) that enables CCR2 to effectively control directional cell migration in different biological contexts. Moreover, we hypothesize that external cues involving ligand concentration and interactions with extracellular matrix components define a switch between a scenario where the receptor rapidly desensitizes and one where it remains responsive, continues to migrate, and efficiently scavenges chemokine. The objective of this proposal is to achieve a comprehensive and predictive systems-based understanding of the signaling and trafficking mechanisms that regulate CCR2 migration and scavenging functions. Here we propose three Aims: (i) We will delineate the roles of key transducers of CCR2- mediated migration and scavenging by combining pharmacological inhibition and gene silencing with cell based assays of migration and scavenging and fluorescence imaging of receptor trafficking. We will also further define the mechanisms that regulate the switch from receptors being desensitized upon activation to maintaining responsiveness and becoming efficient scavengers; (ii) We will discover and characterize novel proteins that regulate CCR2 migration and scavenging using unbiased Mass Spectrometry based approaches coupled with orthogonal methods of analysis and Boolean network modeling to prioritize key regulators for validation as in Aim 1; (iii) We will develop predictive in silico network-based models of CCR2 signaling and trafficking using Boolean reaction-contingency networks initially informed by literature with refinement from systematic data collected in Aims 1 and 2. The expected outcome of this proposal is predictive and spatiotemporally-resolved interactome and signaling network of CCR2 that reveals currently unknown functional and regulatory mechanisms of monocyte migration and scavenging.
Due to its role in regulating the recruitment of monocytes into tissues, the chemokine receptor CCR2 is key to the etiology of many inflammatory diseases and has become an important therapeutic target with ongoing clinical trials. This proposal seeks a comprehensive, predictive, system-level understanding of the temporally- and spatially-resolved CCR2 signaling interactome that controls monocyte function. Such understanding will inform strategies for optimal inhibition of CCR2 by directly targeting the receptor or carefully selected downstream effectors.
