Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells.
OBJECTIVE
The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells.


METHODS
We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies.


RESULTS
Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact.


CONCLUSIONS
We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation.
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Tobias
T
Boothe
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Gareth E
GE
Lim
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Haoning
H
Cen
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Søs
S
Skovsø
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Micah
M
Piske
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Shu Nan
SN
Li
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Ivan R
IR
Nabi
Pôle d'endocrinologie, diabète et nutrition, Institut de recherche expérimentale et clinique, Université catholique de Louvain, Brussels, Belgium.
Patrick
P
Gilon
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada. Electronic address: James.D.Johnson@ubc.ca.
James D
JD
Johnson
Autocrine insulin signaling; Insulin receptor internalization; Insulin resistance; Pancreatic islet beta-cells