Download Summary Cells respond to extracellular cues via receptor signaling

Summary
Cells respond to extracellular cues via receptor signaling. In this manner, cellular behavior is under strict
control of hormones, growth factors or neurotransmitters. Binding of a ligand to its cognate receptor triggers
a cascade of intracellular signaling events. In general, these cascades branch into different series of
molecular conversions leading to, for instance, activation of transcription factors, regulation of metabolic
processes or changes in cell morphology.
To regulate such ‘organized complexity’, molecular interactions in the cell are specific and regulated.
Specificity is guaranteed by recognition domains and regulation of interactions or catalytic conversions
means that they can be ‘swithched on or off’. The latter can be realized, for instance, by a conformational
change that alters the accessibility of an interaction domain.
Intriguingly, most signaling components function in multiple signaling cascades. For example, the >800
members of the G Protein-Coupled Receptor (GPCR) family trigger a vast variety of cellular responses via a
relatively limited set of signaling components. This is achieved by compartmentalization of the signaling
pathways. For example, GPCRs and multiple downstream signaling components are often clustered into
signaling complexes by so-called scaffolding proteins. Such spatial restriction facilitates the coupling of the
receptor
to
its
effectors,
while
it
leaves
other
signaling
compartments
unaffected.
Thus,
compartmentalization confers specificity to signaling cascades. In addition, such spatial organization can
direct signaling activity towards the appropriate subcellular location, depending on the involved process.
Signaling complexes are dynamically regulated, i.e. they can be assembled in response to extracellular
signals. The dynamic targeting of a signaling molecule to a specialized compartment becomes manifest as a
stimulus-induced translocation. In this thesis, we studied the translocations of two proteins: that of
chloride intracellular channel 4 (CLIC4) and that of exchange protein directly activated by cAMP 1 (Epac1).
Central in these studies were the microscopical techniques that allowed visualization of these translocations
with maximal spatiotemporal resolution, most prominently via confocal imaging and measurement of FRET.
Chapter 1 presents a general introduction into the phenomenon of translocations as they occur in cell
signaling. The focus lies on signaling cascades activated by GPCRs since these are most relevant to the
experimental work described in this thesis. Furthermore, this chapter introduces the microscopical
techniques that have been employed to reveal and study ‘our‘ translocations.
In chapter 2, we show that CLIC4 translocation is selectively observed in response to G(α)13-activating
GPCR agonists and requires active RhoA. Contrasting with the ‘membrane insertion model’, the proposed as
activation mechanism for CLIC proteins [1], we show that CLIC4 translocation does not preceed channel
formation in the plasma membrane. Instead, the translocation anchor is the ligand-activated receptor
complex itself. Although we await final proof, our data strongly suggest that recruited CLIC4 plays a
regulatory role in the signal transfer from the stimulated GPCR to RhoA activation. In support of this
hypothesis, CLIC4 has been reported to interact with the scaffold proteins AKAP350 [2] and 14-3-3 [3].
AKAP-Lbc, a family member of AKAP350, serves both as a GPCR scaffold and as a guanine exchange factor
(GEF) coupling G(α)12 to RhoA [4] and is, furthermore, under the regulation of 14-3-3 [5]. Therefore, we
hypothesize that CLIC4 translocates to a signaling compartment containing the activated receptor, G(α)13
and an AKAP scaffold with GEF activity towards RhoA. In this model, the spatial regulation of CLIC4 will
undoubtedly add to the switch-like regulation of RhoA activity.
Chapters 3 and 4 describe two distinct plasma membrane translocations of Epac1. The cAMP effector
Epac1 is a GEF for the GTPase Rap. Interestingly, Rap activity at the plasma membrane induces cell
adhesion. The two types of Epac1 translocation are triggered by cAMP binding and by phosphorylation of
Ezrin/Radixin/Moesin (ERM) proteins, respectively. Binding of cAMP induces a conformational change, that
not only elicits GEF activity by Epac1 [6], but also releases an affinity for the plasma membrane. As a
consequence, Epac1 becomes uniformly distributed along the plasma membrane upon elevated cAMP levels.
In contrast, recruitment of Epac1 by ERM proteins is regulated by ERM phosphorylation. When
phosphorylated, ERM proteins directly bind Epac1, regardless of its activation state, and thereby recruit the
GEF to asymmetrical regions of the plasma membrane.
Since both translocation mechanisms could be specifically disabled by mutagenesis, we were able to
assess their relative importances for the ability of Epac1 to induce Rap-dependent cell adhesion.
Interestingly, we found that the two targeting mechanisms cooperate in this process. We conclude that the
targeting mechanisms dynamically compartmentalize cAMP-Epac1-Rap signaling at the plasma membrane,
thereby enhancing the efficiency and specificity of action.
Besides Epac1 signaling, also the classical cAMP effector PKA is extensively compartmentalized. To date,
more than 50 A kinase anchoring proteins (AKAPs) have been identified. Among the clustered signaling
components are PKA, PKA substrates as well as the enzymes that synthesize cAMP (adenylyl cyclase) and
degrade cAMP (phosphodiesterases) [7,8]. Clearly, to study compartmentalization of the second messenger
cAMP, measurement with spatiotemporal resolution is required. For this, gene-encoded sensors based on
Fluorescence Resonance Energy Transfer (FRET) are indispensible. Chapter 5 describes the development of
CFP-Epac-YFP, an Epac-based FRET-sensor for cAMP. The cAMP-induced conformational change is registered
as a loss of FRET between the terminally fused fluorophores. This novel cAMP sensor has several advantages
over the previously reported PKA-based cAMP sensor. Most importantly, it measures over an increased
dynamic range of cAMP concentrations and displays larger changes in FRET. For the study of cAMP
compartmentalization, the single polypeptide CFP-Epac-YFP can be targeted to specific subcellular
compartments [9], which would be highly complicated (if not impossible) for the two constructs that
constitute the PKA-based sensor.
In chapter 6, we imaged cAMP with spatiotemporal resolution. Not subcellularly though, but
‘intercellularly’. By mixing cells with and without glucagon receptors, we could monitor intercellular transfer
of cAMP through gap junctions after glucagon stimulation. From this, we learned that gap junctional
exchange of cAMP occurs over the entire range of physiological cAMP concentrations and that
phosphodiesterases are the main determinant in controlling the amount of communicated cAMP.
The
studies
presented
in
Chapters
7,
8
and
9
are
not
directly
related
to
the
theme
of
compartmentalization, but have evolved from the previous chapters. Chapter 7 shows that the plasma
membrane lipid PI(4,5)P2 directly regulates communication via connexin43-based gap junctions. The trigger
for gap junctional closure is the PLCβ3-mediated hydrolysis of PI(4,5)P2 itself rather than the concomitantly
produced second messengers diacylglycerol (DAG) or inositoltriphosphate (IP3). Chapter 8 describes the
optimization of the fluorophore pairs used in the Epac-based cAMP sensor. Comparative analyses revealed
optimal donor-acceptor pairs depending on the method employed to measure FRET. For the frequently
applied ratiometric FRET assays, optimal properties are displayed by the CFP-cp173Venus pair. Finally, in
Chapter 9 the Epac1-based FRET sensors were employed to characterize the new Epac1-specific cAMP
analogue 8-pCPT-2’-O-Me-cAMP-AM (also known as 007-AM). The hydrophobic acetoxymethyl (AM) ester
was attached to render the compound more membrane-permeable than the parent compound 007. Once
007-AM passes the plasma membrane, the AM-group will be cleaved by intracellular esterases and as a
consequence the trapped 007 rapidly accumulates in the cytoplasm. Indeed, our sensors demonstrated the
accelerated kinetics and increased efficiency of Epac1 activation in comparison to 007.
In conclusion, heaving read this thesis one may appreciate that the use of sophisticated imaging techniques
and the development of gene-encoded sensors go hand in hand with expanding insights in the
compartmentalization of cell signaling.
References
1.
Harrop S.J., DeMaere M.Z., Fairlie W.D., Reztsova T., Valenzuela S.M., Mazzanti M., Tonini R., Qiu M.R.,
Jankova L., Warton K. et al. (2001). Crystal structure of a soluble form of the intracellular chloride ion
channel CLIC1 (NCC27) at 1.4-A resolution. J. Biol. Chem. 276: 44993-45000.
2.
Shanks R.A., Larocca M.C., Berryman M., Edwards J.C., Urushidani T., Navarre J., and Goldenring J.R.
(2002). AKAP350 at the Golgi apparatus. II. Association of AKAP350 with a novel chloride intracellular
channel (CLIC) family member. J. Biol. Chem. 277: 40973-40980.
3.
Suginta W., Karoulias N., Aitken A., and Ashley R.H. (2001). Chloride intracellular channel protein CLIC4
(p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms.
Biochem J 359: 55-64.
4.
Diviani D., Soderling J., and Scott J.D. (2001). AKAP-Lbc anchors protein kinase A and nucleates Galpha
12-selective Rho-mediated stress fiber formation. J. Biol. Chem. 276: 44247-44257.
5.
Jin J., Smith F.D., Stark C., Wells C.D., Fawcett J.P., Kulkarni S., Metalnikov P., O’Donnell P., Taylor P.,
Taylor L. et al. (2004). Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding
proteins involved in cytoskeletal regulation and cellular organization. Curr. Biol. 14: 1436-1450.
6.
de Rooij J., Rehmann H., van Triest M., Cool R.H., Wittinghofer A., and Bos J.L. (2000). Mechanism of
regulation of the Epac family of cAMP-dependent RapGEFs. J. Biol. Chem. 275: 20829-20836.
7.
Tasken K. and Aandahl E.M. (2004). Localized effects of cAMP mediated by distinct routes of protein
kinase A. Physiol. Rev. 84: 137-167.
8.
Wong W. and Scott J.D. (2004). AKAP signalling complexes: focal points in space and time. Nat. Rev.
Mol. Cell Biol. 5: 959-970.
9.
DiPilato L.M., Cheng X., and Zhang J. (2004). Fluorescent indicators of cAMP and Epac activation reveal
differential dynamics of cAMP signaling within discrete subcellular compartments. Proc. Natl. Acad. Sci. U
S A 101: 16513-16518.