Abstract
In this study, we identified an adhesion-regulated subunit of the interleukin-1 (IL-1) receptor complex. Transfection of fibroblasts with an IL-1 receptor-EGFP construct showed that the fusion protein was located at focal adhesions in cells attaching to fibronectin. Fibronectin attachment caused enhancement in endogenous IL-1 type I receptor levels from on average 2500 to 4300 receptors/cell. In addition, matrix attachment resulted in a decrease in binding affinity (Ka) from 1.0 x 10(9) (M-1) to 5.6 x 10(8) (M-1), due to a 2-fold reduction in association rate constant. The adhesion-mediated effects were reversed by soluble heparin. Cross-linking experiments showed that in cells attached to fibronectin, 50-70% of the radiolabeled IL-1 was associated with a heparinase sensitive, high molecular mass component of about 300 kDa, with a core protein of 80-90 kDa. Formation of the complex was dependent on cell interaction with the heparin binding region in fibronectin and required IL-1/type I IL-1 receptor binding. This report demonstrates the recruitment of a heparan sulfate to the IL-1 receptor complex, following attachment to fibronectin, which correlates with alterations in receptor function. The data suggest that the heparan sulfate constitutes an attachment regulated component of the IL-1 receptor complex with the role of mediating matrix regulation of IL-1 responses.
Highlights
In this study, we identified an adhesion-regulated subunit of the interleukin-1 (IL-1) receptor complex
In this report we demonstrate the presence of a novel, attachment-regulated component of the IL-1 receptor complex at focal adhesions that constitutes a heparan sulfate proteoglycan
Transfection experiments showed the IL-1 receptor protein in adherent cells to be located at sites of focal adhesion, in agreement with earlier reports using radiolabeled ligand [27]
Summary
(Received for publication, November 2, 1998, and in revised form, February 26, 1999). The receptor proximal stages of signal activation involve the adapter proteins TRAF 6 [23] and MyD88 (24 –26) and two related kinases, IRAK-1 and IRAK-2 [7, 24] In adherent cells, such as fibroblasts, IL-1 receptors are located at focal adhesions [27, 28], and IL-1 binding to the type I receptor has rapid effects on cell structure [29]. In this report we demonstrate the presence of a novel, attachment-regulated component of the IL-1 receptor complex at focal adhesions that constitutes a heparan sulfate proteoglycan Recruitment of this component affects receptor function, both in terms of the level of type I IL-1 receptors and their affinity for ligand, and correlates with IL-1-mediated, attachment-regulated signaling and biological responses. The receptor uses a well conserved mechanism for regulating immune and inflammatory responses [3,4,5,6,7] but
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