Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor.
Signals that can mediate ligand-induced receptor internalization and calcium regulation are present in a 48-amino acid "calcium-internalization" domain in the C' terminus of the epidermal growth factor (EGF) receptor. The basis of calcium and internalization regulation signalled by this 48-amino acid sequence was analyzed using deletion and substitution mutant receptors. Cells expressing truncated receptors containing either the NH2- or COOH-terminal portion of the 48-residue domain displayed high affinity EGF-dependent endocytosis and receptor down-regulation. These endocytosis-competent EGF receptor mutants that lacked any autophosphorylation site were unable to increase the concentration of intracellular calcium. To investigate the role of self-phosphorylation in EGF-induced calcium mobilization, phenylalanine was substituted for the single autophosphorylated tyrosine residue in this region of an internalization-competent truncated receptor. The receptor-mediated calcium response was abolished, while ligand-dependent receptor internalization was unimpaired. These results demonstrate that EGF-dependent receptor endocytosis and calcium mobilization are separate events. Tyrosine self-phosphorylation is required for increased [Ca2+]i, while structural features distinct from autophosphorylation are required for receptor internalization.
Chang
C P
CP
Department of Medicine, University of California, San Diego, La Jolla 92093.
Kao
J P
JP
Lazar
C S
CS
Walsh
B J
BJ
Wells
A
A
Wiley
H S
HS
Gill
G N
GN
Rosenfeld
M G
MG
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D015536:Down-Regulation; D004705:Endocytosis; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D010766:Phosphorylation
D008024:Ligands; D066246:ErbB Receptors; D002118:Calcium