Distinct structural specificities for functional coupling of the epidermal growth factor receptor to calcium-signalling versus phospholipase A₂ responses

Activation of phospholipase C (PLC), leading to a rise in cytosolic Ca²⁺, and of phospholipase A₂ (PLA₂), leading to a release of arachidonic acid, are among the early transmembrane signalling events that have been demonstrated in response to occupancy of the epidermal growth factor (EGF) receptor. The tyrosine kinase activity of the receptor has been shown to be necessary for both of these responses. This requirement for the tyrosine kinase activity could conceivably implicate a role for receptor autophosphorylation in the activation of PLA₂. We now demonstrate that coupling of the EGF receptor to PLA₂ was not impaired in a deletion mutant (CD126) devoid of the 126 amino acids from the C-terminus which include four major autophosphorylation sites. Functional coupling of the EGF receptor to PLA₂ was demonstrated using three different experimental designs: (1) release of [¹⁴C]arachidonic acid from prelabelled intact cells, (2) release of [³H]arachidonic acid from prelabelled cells permeabilized with glass beads, and (3) direct measurement of PLA₂ enzymic activity in cell-free extracts using an 'in vitro' assay employing exogenous phospholipid substrate. Functional coupling of the EGF receptor to PLA₂ occurred despite the absence of a demonstrable Ca²⁺-signalling response and the detection of diminished but persistent PLC-γ phosphorylation on tyrosine residues in the CD126 deletion mutants. These results point to a clear distinction in the biochemical mechanism and role for receptor autophosphorylation in functional coupling of the EGF receptor to PLA₂ activation versus Ca²⁺ signalling.