Dissecting Discoidin Domain Receptor 2 Dynamics in Fibrosis with a Programmable Collagen-Mimetic Probe

Collagen receptors orchestrate vital extracellular matrix signaling, yet the inability to distinguish functional receptor activation from mere expression in real-time has obscured our understanding of fibrotic progression. Here, we develop a fluorescent triple-helical collagen-mimetic peptide (CMP) probe, [GVMGFO]3, designed to selectively target the ligand-engaging conformation of Discoidin Domain Receptor 2 (DDR2)─the sole receptor tyrosine kinase family that signals collagen. In pulmonary fibrosis, this probe identifies active fibrotic niches in vivo and directly tethers to disease-driving activated fibroblasts to enable precise ex vivo mapping, all without perturbing baseline signaling. Utilizing this tool, we decipher the long-standing mystery of DDR2’s characteristically slow, hours-long activation kinetics. We reveal that while monomeric collagen engagement fails to override constitutive DDR2 internalization, supramolecular fibrillar collagen provides a multivalent physical anchor that arrests DDR2 trafficking at the cell–matrix interface to sustain receptor clustering and phosphorylation. To recapitulate this biophysical requirement, we engineered Zn2+-coordinated supramolecular assemblies of a histidine-modified CMP, [H-GVMGFO-H]3, triggering rapid DDR2 activation within minutes. Our work transforms CMPs from structural models of collagen into programmable chemical tools for dissecting the spatiotemporal dynamics of collagen–receptor interplay, offering a platform for imaging and modulating fibrotic disease.