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

Abstract

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.

Membrane-permeable Calmodulin Inhibitors (e.g. W-7/W-13) Bind to Membranes, Changing the Electrostatic Surface Potential

“…Villalobo and co-workers (18,20) showed that EGFR is a Ca 2ϩ / CaM-binding protein. What is the physiological significance of this binding in EGF-mediated activation of EGFR?…”

“…Although one must obviously be cautious when interpreting cell biological experiments with CaM inhibitors, we do wish to suggest that they can provide useful information about cellular processes. For example, Ca 2ϩ /CaM binds to the EGFR (18,20) and to peptides corresponding to its JM region (19,21,53). Does this binding contribute to the activation of the receptor?…”

Intracellular Transactivation of Epidermal Growth Factor Receptor byα1A-Adrenoceptor Is Mediated by Phosphatidylinositol 3-Kinase Independently of Activation of Extracellular Signal Regulated Kinases 1/2 and Serine-Threonine Kinases in Chinese Hamster Ovary Cells

“…All three a 1 -AR subtypes couple to G q and result in activation of phospholipase C with an increase in inositol triphosphate and subsequent mobilization of intracellular Ca 21 , which leads to activation of the calcium/calmodulin (Ca 21 /CaM) pathway (Wu et al, 1992;Bylund et al, 1994;Della Rocca et al, 1997;Koshimizu et al, 2003). The Ca 21 /CaM pathway directly and indirectly influences EGFR and has a regulatory function in EGFR signaling (Murasawa et al, 1998;Aifa et al, 2002;Li and Villalobo, 2002;Sanchez-Gonzalez et al, 2010). Other important signaling molecules activated by a 1 -AR are the Src (Han et al, 2008) and PI3K, which are also implicated in EGFR transactivation.…”

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

“…EGFR signalling induces an early and transient increase in the cytosolic concentration of free Ca 2 + [14][15][16], which results in the formation of the Ca 2 + -CaM complex. We have previously shown that the Ca 2 + -CaM complex binds to the EGFR in vitro and in living cells [17][18][19][20][21][22][23]. The CaM-binding domain (CaM-BD) of the receptor is located at its cytosolic juxtamembrane region [19,[23][24][25] and binding of Ca 2 + -CaM to this site exerts a positive triggering role in the ligand-dependent activation of the EGFR in living cells [21][22][23]25].…”