Research projects
Functional Proteomics of Signal Transduction
by Membrane Receptors
One of the most challenging endeavours of
modern biological science is the current effort to understand
the function of living cells at the molecular level, i.e. how
do cells achieve finely tuned control of growth and division,
metabolism, shape and movement in response to dynamic internal
and external cues. Despite an unprecedented proliferation in the
quantity and quality of the available information, large gaps
in our knowledge still exist. The introduction of highly-parallel
technologies embodied in various "omics" approaches
has begun to give a more global view of cellular signalling and
response systems, but at the same time has demonstrated that these
cellular systems are much more complicated than initially anticipated.
Many cellular functions depend on various kinds of receptor-mediated
signalling. The G-protein-coupled receptors (GPCRs) are one of
the largest protein superfamilies in the human genome and constitute
a major proportion of cellular receptors, but at present many
GPCRs are orphans, i.e. the relevant physiological ligand is unknown.
GPCRs link the cell to its environment by receiving stimuli, transmitting
this signal to the cell, and initiating and regulating the response.
It is no surprise that these receptors are involved in numerous
disorders and that, with kinases, they predominate as targets
for development of drugs.
The classical paradigm for GPCR signalling
was essentially linear and sequential. However, more recent evidence
shows that the signalling pathways of GPCRs are very complex.
Both classical GPCR pathways and G-protein-independent pathways
can be activated and responses can involve cross-regulation of
many specific pathways, including cross-talk between different
GPCRs as well as interactions with other signaling pathways. Interactions
with adaptor and scaffolding proteins can enhance the activation
of proteins in the signal cascades, ensure specificity by bringing
only the necessary proteins into close proximity, and effect a
compartmentalization of the signalling cascade. GPCRs may undergo
dimerization and even higher order oligomer formation, which can
contribute to the diversity of signalling by altering the specificity
of agonist and antagonist interactions. Although the evidence
is inconclusive at present, there are increasing indications that
in a single cell different molecules of a given GPCR may contribute
in different ways to the cellular response upon stimulation.
Many aspects of cellular life are regulated
via protein phosphorylation. Phosphorylation plays a major role
in intracellular communication during development, in physiological
responses, in homeostasis and in the functioning of the nervous
and immune systems. This very sophisticated cellular regulatory
mechanism involves phosphorylation/ dephosphorylation of as much
as 30% of all cellular proteins, including of the GPCR superfamily.
Stimulation of GPCRs typically triggers a cascade of phosphorylation
events on other cellular proteins, which in turn feedback to modifications
of GPCRs that are critical to their functional roles. In the companion
to this paper, we present evidence from studies of rapid changes
in the cellular phosphoproteome that ETA and ETB receptors engage
a wide variety of cellular signalling pathways upon stimulation
of human lung fibroblasts.
Given their low cellular abundance and status
as integral membrane proteins, direct characterization of post-translational
modifications of GPCRs has been limited. Apart from pioneering
work on rhodopsin, there are still only a few scattered reports
in the literature involving direct observation by mass spectrometry
of post-translational modifications of GPCRs. Much of what is
presently known about post-translational modification of GPCRs
is largely based on indirect methods. These include genetic substitution
of amino acid residues that are possible sites of modification
(e.g., Ser, Thr, Tyr for phosphorylation or Cys for acylation)
and the in vitro phosphorylation/dephosphorylation by kinases
or phosphatases of synthetic peptides derived from the receptor
amino acid sequences. There are indications that these types of
indirect studies may give misleading results and in some cases
it has been observed that genetic substitutions, e.g. Ser to Ala,
can lead to phosphorylation of alternative residues in protein
sequences with at most very moderate influences on protein activity.
We have developed methods for rapid, mild
isolation of membrane receptors which, combined with the high
sensitivity of MALDI and electrospray ion trap mass spectrometry,
greatly facilitate the direct evaluation of the sites and types
of post-translational modifications of GCPRs. The present investigations
of the post-translational modifications of ETA and ETB receptors
of human lung fibroblasts both prior and subsequent to stimulation
reveal an unsuspected number of isoforms involving differently
post-translationally modified receptors. We suggest that the multiple
phenotypes observed for individual protein sequences may correspond
to another level of diversification in the function of endothelin
receptors, and that this is coupled to the wide diversity in the
types of responses elicited by endothelin stimulation.
Functional Diversity of Endothelin Pathways
in Human Lung Fibroblasts May be Based on Structural Diversity
of the Endothelin Receptors
Post-translational modifications of the endothelin
receptors A and B from human lung fibroblasts were investigated
before and after stimulation of the cells with (dA)30-5'-S-EMC-Endothelin-1.
The patterns of phosphorylation and palmitoylation of both receptors
were much more complicated than expected. In both the stimulated
and unstimulated states, multiple isoforms differing in the number
and location of post-translational modifications were present.
MS analyses suggested rapid changes in these isoforms following
stimulation. Overall, ETA receptor was modified at 20 sites (15
phosphorylation, 5 palmitoylation sites) and ETB at 17 sites (13
phosphorylation, 4 palmitoylation sites). Part of the structural
diversity involved hyper-modification of short sequence regions
and it is suggested that this could represent a mechanism for
incremental modulation of receptor activity. It is postulated
that the observed structural diversity over disparate parts of
the receptor sequences forms the basis for parallel stimulation
of different signalling pathways at spatially and functionally
distinct ET receptors differing in post-translational modifications.
Rapid Changes in the Phosphoproteome Show
Diverse Cellular Responses
Following Stimulation of Human Lung Fibroblasts with Endothelin-1
The rapid phosphorylation and dephosphorylation
of a variety of proteins downstream of the endothelin receptors
A and B was investigated following stimulation of human lung fibroblasts
with endothelin-1. Changes in the phosphorylation of proteins
involved in the cell cycle, the cytoskeleton, membrane channels,
transcription, angiogenesis and metabolism were observed. From
observed changes in Protein Phosphatase 2A, CDC25 A, and Caspase-2
precursor, a model for the promotion of cell cycle progression
by ET-1 stimulation is proposed. This may offer insights into
the mechanisms by which ET-1 exerts its mitogenic effects. The
identities of the other proteins phosphorylated within two minutes
of stimulation indicate that endothelin-1 also rapidly engages
a diverse variety of other cellular responses.
Phosphoproteomics of Cystic Fibrosis
Cystic fibrosis (CF) is a recessive genetic
disorder, mainly caused by mutations in the gene that codes for
the cystic fibrosis transmembrane conductance regulator (CFTR).
CFTR is an integral membrane protein and belongs to the superfamily
of ATP-binding cassette (ABC) transporters. CFTR mainly acts as
a chloride channel, but is also able to transport a variety of
other molecules like reduced and oxidised glutathione.
CFTR functions in macromolecular complexes, is regulated by PKA
and PKC phosphorylation and itself influences the function of
other ion channels in the plasma membrane. Reliable protein markers
for diagnosis and to follow the course of the disease are virtually
absent and the knowledge about signalling events downstream of
CFTR after activation is rudimentary.
In different top-down functional proteomics
approaches we firstly analyse the influence of the most common
CFTR mutation (DF508) on the phosphoproteome of affected cells
with the aim to find biomarkers for the disease. Secondly, we
track signalling events downstream of CFTR after stimulation of
cells with cyclic-AMP to find out how the protein influences the
function of other proteins and to identify potential new functions.
The bottom-up approaches include the time depended analysis of
in vivo phosphorylation processes on CFTR and the search for direct
protein-interaction partners.
For the described approaches we developed new and also use long
established CF related cell models.
Global Observation of Post-Translational Modifications
We have developed method involving enrichment
and fractionation of intact, phosphorylated proteins. Immobilized
metal ion affinity columns optimized for phos-phoproteins and
immobilized antibody columns specific for phospho-(Ser, Thr, Tyr)
residues were combined. This allowed very strong enrichment of
phosphoproteins and the selection of phos-phoproteins with specific
types of phosphorylation, e.g. proteins phosphorylated only on
one of Ser, Thr or Tyr. It has been reported that this allows
observation on 2D gels of hundreds of low abundance proteins that
are subject to phosphorylation as a consequence of cellular stimulation
with endothelin, platelet derived growth factor or epithelial
growth factor.
Functional Phosphoproteomics and Targeting
Signalling Networks
In collaboration with ProteoSys, AG, Germany
we have developed Proteo-Mode, an instrument for automated, high
throughput preparation of phosphoproteins for proteomics analysis
of complex cellular signaling networks involving multiple, time
dependent protein phosphorylation events. This enables the integrated
response of complex cellular signaling networks to be analysed
in normal and abnormal (disease) states and provides new perspectives
in targeting and evaluation of the effects of therapeutic compounds
on such networks.
Ultra-High Sensitivity Multi-Photon Imaging
and Quantitation in Proteomics Analysis
Multi-photon-detection (MPD) is a method for
the measurement of radioisotopes which decay by the electron capture
mechanism and hence emit multiple photons/particles essentially
simultaneously. Background radioactivity very rarely provides
coincident emissions of defined energies and can therefore be
rejected from the analysis. Coincidence detection of multiple
emissions by MPD thus permits exquisitely sensitive detection
for radioactivity levels well below background radiation levels.
Since single decay events are detected, in principle MPD imagers
can show linear signals over eight orders of magnitude down to
about 600 molecules).
This project is carried out in collaboration with Dr A. Drukier,
Biotraces, Inc. USA
New Paradigms in Cellular Function and Top-Down
Proteomics Analyses
New paradigms suggest that many aspects of
cellular function are controlled by rapid, stochastic, combinatorial
processes. The implications of these new paradigms for proteomics
are considered in relation to the information content of top-down
and bottom-up proteomics approaches. Functional evidence and current
proteomics experiments suggest that phenotypic variation of individual
proteins is a major form of control for the combinatorial processes
and that in many cases top-down proteomics approaches will be
essential for proteomics investigations of cellular function.
It is suggested that the new paradigms and the nature of the general
cellular processes that affect protein phenotypes should be taken
into account in the development of proteomics methodology. More
generally, the coupling of stochastic, combinatorial processes
to energy-dependent processes that change protein phenotypes may
represent one of the basic principles of cellular function.
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this page is always under construction by J. Godovac-Zimmermann
last update: February 17th,
2005