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New Biological Frontiers Illuminated by Molecular Sensors and Actuators
Poster Abstracts
17-POS
Board 17
Insulin Receptor and IGF1 Receptor Biosensors Employing Between-Domain Fluorescence
Tags
James D. Johnson
, Howard Cen, Søs Skovsø, Tobias Albrecht.
University of British Columbia, Vancouver, Canada.
The insulin receptor (InsR) is one of the most studied proteins, but fundamental aspects of its
biology remain unclear, including the kinetics of insulin receptor activation and internalization
from the plasma membrane. To monitor insulin receptor and insulin-like growth factor 1 receptor
(IGF1R) dynamics in real time, we employed a novel tagging strategy wherein fluorescent
proteins (TagRFP, TagRFP-T, eGFP, eYFP, TagBFP, tagGFP2) were placed at the extracellular
region in between two functional domains. Unlike published insulin receptor fusions that have
fluorescent proteins attached to their C-termini, our fusion proteins primarily localized to
vesicle-like cytoplasmic structures mirroring the pattern we observed for the endogenous insulin
receptors in vivo and in vitro. InsRA and InsRB splice variants had overlapping distributions, in
contrasting with the conclusions of previous studies with C-terminal tagged receptors. Using
insulin secreting pancreatic beta-cells as a model system, we mapped the steady state
internalization and trafficking of between-domain-tagged InsRs and endogenous InsRs to
organelles labeled with caveolin1 (Cav1), flotillin1 and Lamp1, which bypass compartments
positive for clathrin, Rab5a, Rab7, Rab11a, or Rab4a. TIRF imaging of InsRA-eGFP revealed
that the phospho-mimetic Cav1-Y14D mutant significantly shortened the lifetime of InsRA
domains at the plasma membrane prior internalization. Ratiometric pH sensor fluorescent
proteins and Fluorogen Activating Peptides were also developed to track the dynamics of insulin
receptor internalization of insulin receptor clusters in living cells in real time using spinning disk
confocal microscopy and high-conetnt imaging systems. We expect that these novel approaches
to live-cell imaging of tyrosine kinase receptor dynamics will be important for elucidation of
insulin and IGF1 signaling and development of high-content screening for molecules that modify
insulin receptor trafficking and activity.