Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance.
One early response to epidermal growth factor receptor (EGFR) activation is an increase in intracellular calcium. We have used surface plasmon resonance (SPR) to study real-time interactions between the intracellular juxtamembrane (JM) region of EGFR and calmodulin. The EGFR-JM (Met(644)-Phe(688)) was expressed as a GST fusion protein and immobilised on a sensor chip surface. Calmodulin specifically interacts with EGFR-JM in a calcium-dependent manner with a high on and high off rate. Chemical modification of EGFR-JM by using arginine-selective phenylglyoxal or deletion of the basic segment Arg(645)-Arg(657) inhibits the interaction. Phosphorylation of EGFR-JM by protein kinase C (PKC) or glutamate substitution of Thr(654) inhibits the interaction, suggesting that PKC phosphorylation electrostatically interferes with calmodulin binding to basic arginine residues. Calmodulin binding was also inhibited by suramin. Our results suggest that EGFR-JM is essential for epidermal growth factor (EGF)-mediated calcium-calmodulin signalling and for signal integration between other signalling pathways.
D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010658:Phenylglyoxal / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D020349:Surface Plasmon Resonance
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D066246:ErbB Receptors; D011493:Protein Kinase C; D010658:Phenylglyoxal