Downregulation of EGFR Signaling Requires Gαs

We previously found that Gαs promotes EGF‐induced EGFR degradation (Zheng, et al. MBC 2004, 15:5538). Since degradation is key step in the downregulation of EGFR signaling, we predict that Gαs facilitates signal downregulation. To determine if this is the case, control, Gαs, or Gαi3 depleted HeLa cells stimulated with EGF were monitored for EGFR autophosphosphorylation by immunoblotting using tyrosine site‐specific antibodies (pY1045, Y1068). We found that Gαs but not Gαi3 depletion increases EGFR autophosphorylation at 5 and 15 min after EGF stimulation, and the effects of Gαs depletion were partially rescued by transfection with siRNA resistant Gαs. These data indicate that Gαs is specifically required to downregulate EGFR autophosphorylation. Delayed downregulation and degradation could result from disrupted EGFR trafficking. To determine the effect(s) of Gαs on EGFR trafficking, we monitored the distribution of 647‐EGF and EGFR (pY1068) in control, Gαs, or Gαi3 depleted HeLa cells by immunofluoresence. At 5 min after stimulation, we found that EGFR and 647‐EGF localize to EEA1 and Hrs labeled endosomes in control cells. In contrast, more EGFR and 647‐EGF are found near the PM and show less colocalization in Gαs depleted cells. At 15 min after stimulation, pY1068 and 647‐EGF colocalize with EEA1 and Hrs in control and Gαs depleted cells, indicating that trafficking of EGFR from the PM to endosomes is delayed in the absence of Gαs, which may result in delayed receptor degradation. Overall, our novel data suggest that Gαs affects the downregulation of EGFR signaling by modulating an early (e.g., internalization and/or recycling) step in receptor trafficking.