Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor.
Previous work from our laboratory has demonstrated that the Ca(2+)-calmodulin complex inhibits the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and that the receptor can be isolated by Ca(2+)-dependent calmodulin-affinity chromatography [San José, Bengurija, 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 Ca(2+)-dependent manner when this segment forms part of a recombinant fusion protein [Martijn-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 Ca(2+), 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 Ca(2+)-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. 0.2-0.3 microM. 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.
Li
Hongbing
H
Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientijficas and Universidad Autónoma de Madrid, c/Arturo Duperier 4, E-28029 Madrid, Spain.
Villalobo
Antonio
A
D000469:Alkaline Phosphatase / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D002294:Carcinoma, Squamous Cell; D003432:Cross-Linking Reagents; D004533:Egtazic Acid / Q000494:pharmacology; D066246:ErbB Receptors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D004533:Egtazic Acid; D066246:ErbB Receptors; D000469:Alkaline Phosphatase; D002118:Calcium