Utilization of a receptor reserve for effective amplification of mitogenicsignaling by an epidermal growth factor mutant deficient in receptor activation

The idea of a receptor reserve in mediating cellular function is well known but direct biochemical evidence has not been easy to obtain. This study st ems from our results showing that L15 of epidermal growth factor (ECF) is i mportant in both EGF receptor (EGFR) binding and activation, and the L15A a nalog of human EGF (hEGF) partially uncouples EGFR binding from EGFR activa tion (Nandagopal et al., [1996] Protein Engng 9:781-788). We address the ce llular mechanism of mitogenic signal amplification by EGFR tyrosine kinase in response to L15A hEGF. L15A is partially impaired in receptor dimerizati on, shown by chemical cross-linking and allosteric activation of EGFR in a substrate phosphorylation assay. Immunoprecipitation experiments reveal, ho wever, that L15A can induce EGFR autophosphorylation in intact murine kerat inocytes by utilizing spare receptors, the ratio of total phosphotyrosine c ontent per receptor being significantly lower than that elicited by wild-ty pe. This direct biochemical evidence, based on function, of utilization of a receptor reserve for kinase stimulation suggests that an EGF variant can activate varying receptor numbers to generate the same effective response. L15A-activated receptors can stimulate mitogen-activated protein kinase (MA PK) that is important for mitogenesis. The lack of linear correlation betwe en levels of receptor dimerization, autophosphorylation, and MAPK activatio n suggests that signal amplification is mediated by cooperative effects. Fl ow cytometric analyses show that the percentages of cells which proliferate in response to 1 nM L15A and their rate of entry into S-phase are both dec reased relative to I nM wild-type, indicating that MAPK activation alone is insufficient for maximal stimulation of mitogenesis. Higher concentrations of L15A reverse this effect, indicating that L15A and wild-type differ in the number of receptors each activates to induce the threshold response, wh ich may be attained by cooperative activation of receptor dimers/oligomers by van der Waal's weak forces of attraction. The maintenance of a receptor reserve underscores an effective strategy in cell survival. Published 2001 Wiley-Liss, Inc.