Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor

Previous work from our laboratory has demonstrated that the Ca2+—calmodulin complex inhibits the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and that the receptor can be isolated by Ca2+-dependent calmodulin-affinity chromatography [San José, Benguría, Geller and Villalobo (1992) J. Biol. Chem. 267, 15237–15245]. Moreover, we have demonstrated that the cytosolic juxtamembrane region of the human receptor (residues 645–660) binds calmodulin in a Ca2+-dependent manner when this segment forms part of a recombinant fusion protein [Martín-Nieto and Villalobo (1998) Biochemistry 37, 227–236]. However, demonstration of the direct interaction between calmodulin and the whole receptor has remained elusive. In this work, we show that calmodulin, in the presence of Ca2+, forms part of a high-molecular-mass complex built upon covalent cross-linkage of the human EGFR immunoprecipitated from two cell lines overexpressing this receptor. Although several calmodulin-binding proteins co-immunoprecipitated with the EGFR, suggesting that they interact with the receptor, we demonstrated using overlay techniques that biotinylated calmodulin binds directly to the receptor in a Ca2+-dependent manner without the mediation of any adaptor calmodulin-binding protein. Calmodulin binds to the EGFR with an apparent dissociation constant (K′d) of approx. 0.2–0.3μM. Treatment of cells with epidermal growth factor, or with inhibitors of protein kinase C and calmodulin-dependent protein kinase II, or treatment of the immunoprecipitated receptor with alkaline phosphatase, does not significantly affect the binding of biotinylated calmodulin to the receptor.