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Biochemical Society Transactions (1999) 27
Internalisation of Wild type and Mutant als-Adrenergic
Receptors
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Patricia A. Stevens and Graeme Milligan
Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
Davidson Building, University of Glasgow, Glasgow, G 12 8QQ.
Fusion constructs were generated between the wild type,
constitutively active or phosphorylation-negativemutants of the
aIB -adrenergic receptor and green fluorescent protein (GFP).
After stable expression in HEK 293 cells and pharmacological
characterisation, fluorescent confocal microscopy was used to
investigate the internalisation pathways used by this receptor and
to compare the extent of ligand-induced internalisation of the wild
type or mutant alB -adrenergic receptors. Parallel cell surface
biotin labelling experiments allowed the level of internalisation in
each case to be quantified.
G protein activation and effector regulation by the
human S - H T ~ Areceptor and the a subunit of Gil fusion
proteins.
Elaine Kellett, I. Craig Cam and Graeme Milligan
Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
Davidson Building, University of Glasgow, Glasgow, GI2 8QQ.
Fusion proteins were generated between the human 5-HT I A
receptor and both wild type and pertussis toxin resistant forms of
the a subunit of the G protein Gi 1. These, as well as the isolated 5H T ~ Areceptor, were expressed stably in HEK293 cells. The
activation of Gil in these cell lines was compared via 5-HTmediated stimulation of high affinity GTPase activity in the
presence or absence of pertussis toxin. Pertussis toxin treatment
attenuated the GTPase activity in cells expressing the 5-HT1 A
receptor alone. In the cells expressing the 5-HT 1A receptor-wild
type G i l a construct the agonist effect was not fully attenuated by
treatment with toxin although basal GTPase activity was reduced.
The toxin showed no effect on the cells expressing the ~ - H T I A
receptor-(cys35lgIy)~iI a fusion protein.
The regulation of forskolin stimulated adenylyl cyclase activity was
also studied. Low concentrations of 5-HT added to intact cells
expressing the ~ - H T ~receptor
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alone resulted in strong inhibition
of forskolin-amplified adenylyl cyclase and this was partially
reversed at high agonist concentrations. This was more easily
observed in cells treated with pertussis toxin. In the cells expressing
the fusion proteins agonist-mediated inhibition of adenylyl cyclase
was seen but there was no stimulatory element.
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Receptor-GFP Fusion Proteins; A study of Drug Effects
on Receptor Internalisation, Trafficking and Expression
Alison J. McLean and Graeme Milligan
Molecular Pharmacology group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow GI2 8QQ.
Tagging of signalling molecules with green fluorescent protein
(GFP), isolated from the jellyfish Aequoreu victoria, has produced
useful tools in the study of receptor sequestration and trafficking.
We have used this technique previously to study a GFP-tagged
TRH receptor construct [I]. In this study we have linked various
N-terminally Flag tagged beta-adrenergic receptors with GFP at the
C-terminus, and stably transfected them into HEK 293 cells. All
constructs arc functional with the ability to stimulate adenylyl
cyclase. The auto fluorescence of GFP has been used as an
indicator of the location and expression of these receptor-GFP
fusion proteins in response to various drug treatments. This in
conjunction with intact cell radioligand binding and immunological
analysis will help increase understanding of beta-adrenergic
receptor trafficking.
References
[ I ] Drmota, T., Could, G. W. and Milligan, G. (1988). 1. Biol.
Chem. 273,24000-24008.
Cell Cycle Regulation in Rat 1 Fibroblasts Expressing
a Murine Delta Opioid Gi Linked Receptor
*Moira A. Wilson, ?Neil G. Anderson and *Graeme Milligan
*Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical Sciences,
University of Glasgow, Glasgow G I 2 8QQ
?Department of Surgery, Stopford Building, University of
Manchester, Manchester M I3
The transition of mammalian cells from quiescence (Go) to the
first stage of the cell cycle (GI) relies on the transduction of
signals from the extracellular environment to the cell cycle
machinery. Using rat-I fibroblasts stably transfected to express the
murine delta opioid Gi linked receptor, we have looked at the
contribution of the mitogenic pathways activated via this receptor
in control of the cell cycle machinery. Cell cycle progression is
controlled by cyclins and cyclin dependent kinases (CDKs), and
opposed by CDK inhibitors, to drive cells from Go to G I and
through the subsequent stages of the cell cycle. Molecular events
which mark S-phase entry include Rb phosphorylation and
decrease in the levels of the CDK inhibitor p27kiPl.Upon
activation of this receptor we observed a time dependent decrease
in p27kiPl levels and an increase in Rb phosphorylation.
Inhibition of PI 3 kinase and p70 S6 kinase activation had similar
effects in that both had a partial effect on blocking agonist induced
alterations in p27kiPl levels yet had little effect on Rb
phosphorylation. Such data would suggest that upon activation of
this receptor, in these cells,p the PI 3 kinase / p70 S6 kinase
pathway has only a partial input into the mitogenic effects
stimulated via this Gi linked receptor, whereas activation of the
MAPK pathway is critical.