Phosphorylation of the MAPK isoform ERK by G protein-coupled receptors invo
lves multiple signaling pathways. One of these pathways entails growth fact
or receptor transactivation followed by ERK activation. This study demonstr
ates that a similar signaling pathway is used by the mu -opioid receptor (M
OR) expressed in HEK293 cells and involves calmodulin (CaM). Stimulation of
MOR resulted in both epidermal growth factor receptor (EGFR) and ERK phosp
horylation. Data obtained with inhibitors of EGFR Tyr kinase and membrane m
etalloproteases support an intermediate role of EGFR activation, involving
release of endogenous membrane-bound epidermal growth factor. Previous stud
ies had demonstrated a role for CaM in opioid signaling based on direct CaM
binding to MOR. To test whether CaM contributes to EGFR transactivation an
d ERK phosphorylation by MOR, we compared wild-type MOR with mutant K273A M
OR, which binds CaM poorly, but couples normally to G proteins. Stimulation
of K273A MOR with [D-Ala(2), MePhe(4), Gly-ol(5)]enkephalin (10-100 nM) re
sulted in significantly reduced ERK phosphorylation. Furthermore, wild-type
MOR stimulated EGFR Tyr phosphorylation 3-fold more than K273A MOR, indica
ting that direct CaM-MOR interaction plays a key role in the transactivatio
n process. Inhibitors of CaM and protein kinase C also attenuated [D-Ala(2)
, MePhe(4), Glyol(5)]enkephalin-induced EGFR transactivation in wildtype (b
ut not mutant) MOR-expressing cells. This novel pathway of EGFR transactiva
tion may be shared by other G protein-coupled receptors shown to interact w
ith CaM.