Identification of invariant chain CD74 as a functional receptor of tissue inhibitor of metalloproteinases-1 (TIMP-1).

Abstract Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising anti-proteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging pro-inflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta-chain-associated protein kinase-70 (ZAP-70) signalling-associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor (MIF). Here, we demonstrate TIMP-1/CD74 interaction by co-immunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the N-terminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1/CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine binding site of CD74 using a synthetic peptide abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and anti-proteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1/CD74 interaction may shed new light on clinical attempts to therapeutically target ligand-induced CD74 activity in cancer and other inflammatory diseases.