Download Receptor protein tyrosine phosphatase μ

A. Radu Aricescu, Christian Siebold and E. Yvonne Jones1
Cancer Research UK Receptor Structure Research Group, University of Oxford, Henry Wellcome Building of Genomic Medicine, Division of Structural Biology,
Roosevelt Drive, Oxford OX3 7BN, U.K.
Abstract
We review here recent results on the structure and function of a receptor protein tyrosine phosphatase,
RPTPμ. In addition to their intercellular catalytic domains which bear the phosphatase activity, the RPTPs
are cell-surface-receptor-type molecules and in many cases have large extracellular regions. What role
can these extracellular regions play in function? For RPTPμ, the extracellular region is known to mediate
homophilic adhesion. Sequence analysis indicates that it comprises six domains: an N-terminal MAM
(meprin/A5/μ), one immunoglobulin-like domain and four fibronectin type III (FN) repeats. We have
determined the crystal structure of the entire extracellular region for RPTPμ in the form of a functional
adhesion dimer. The physical characteristics and dimensions of the adhesion dimer suggest a mechanism
by which the location of this phosphatase can be influenced by cell–cell spacings.
The receptor protein tyrosine
phosphatases
The RPTPs (receptor protein tyrosine phosphatases) are a
family of cell-surface receptors numbering some 20 members in the human genome [1,2]. Each RPTP has a single
membrane-spanning sequence connecting an N-terminal
ectodomain to a C-terminal cytoplasmic region. The domain
architecture and phosphatase activity of the cytoplasmic
region provides unifying features, common to all family
members (most of them have a tandem of phosphatase domains, but some RPTPs have just one). In contrast, the
extracellular regions of these cell-surface glycoproteins are
very varied. What is the role of the extracellular region in the
function of these molecules and how might it impact on
the activity of the tyrosine phosphatase catalytic domain
within the cell? In the cellular context, a protein, either cytosolic or at the cell surface, is part of a very crowded population
of molecules [3,4]. To fulfil their disparate functions, many
such molecules will need to find their way to particular
subcellular locations. Might this be the role of the extracellular
region of the RPTPs, to determine the subcellular positioning
of the cytoplasmic phosphatase activity? Our recent data
indicate that this could indeed be a function of the
extracellular region for at least one of the RPTPs, providing
an example of the interplay between cell-surface adhesion and
modulation of intracellular signalling [5]. The conclusions we
review here can be paraphrased as answers to the questions
of how an RPTP can ‘measure’ its correct location and how,
Key words: cell adhesion, extracellular complex, homodimer, protein crystallography, receptor
protein tyrosine phosphatase.
Abbreviations used: EM, electron microscopy; FN, fibronectin type III; GFP, green fluorescent
protein; HEK-293, human embryonic kidney; MAD, multiple anomalous dispersion; MAM,
meprin/A5/μ; RPTP, receptor protein tyrosine phosphatase.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2008) 36, 167–172; doi:10.1042/BST0360167
having identified this location, the RPTP can be tethered
to stay there. The RPTP we have focused on is RPTPμ [6],
one of the type IIb subfamily, and, for this particular system,
the correct location is represented by regions of intercellular
contact such as the adherens junctions [7,8].
The extracellular regions of the type IIb RPTPs (RPTPμ,
RPTPρ, RPTPκ and RPTPλ) share a common, six-domain,
architecture and are believed to mediate cellular adhesion by
homodimerization [9–12]. RPTPμ is a cell-adhesion molecule
involved in neural development (axon growth) and angiogenesis [7,13]. Expression is developmentally regulated within
the central nervous system and the vascular network with
clustering of RPTPμ molecules apparent on axonal growth
cones and at endothelial cell junctions [7,14,15]. RPTPμ is
believed to modulate axon growth and angiogenesis not only
through homophilic adhesion, but also through interactions
with members of the cadherin family of adhesion molecules
[16–18]. One of the central questions for the function of
RPTPμ is the nature of its interactions with the cadherins.
Cadherins are themselves homophilic adhesion molecules
and are central to the development and organization of many
tissues within the body [19]. Thus members of the RPTP
type IIb subfamily, such as RPTPμ, may make key contributions to the balance between cellular mobility and adhesion
through the modulation of cadherin function, but how?
Much is known about structure–function relationships,
and the details of the catalytic mechanism, for the cytosolic
regions of the RPTPs; their catalytic domains conform to the
classic phosphatase architecture [20]. An understanding of
the structure–function relationship of the extracellular
regions has remained rather more obscure. For the ectodomains of the type IIb RPTPs, sequence analysis predicted
a MAM (meprin/A5/μ) domain at the N-terminus, followed
by an Ig-like domain and four fibronectin type III (FN)
domains before the single membrane-spanning helix. These
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Receptor protein tyrosine phosphatase μ:
measuring where to stick
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Biochemical Society Transactions (2008) Volume 36, part 2
six domains mediate the homophilic adhesive activity [21]
and formed the focus of our structural studies on RPTPμ.
Structure of the MAM–Ig domains
Eukaryotic cell-based expression was essential for providing
constructs derived from the RPTPμ extracellular region
and suitable for structural studies. We have developed a
robust and efficient protocol for the use of HEK-293T
(human embryonic kidney) cells for transient expression
of soluble secreted proteins [22]. This methodology allows
manipulation of the glycosylation state of a secreted protein;
a functionality that is frequently essential for the finetuning of the homogeneity of glycoproteins to permit the
growth of well-ordered protein crystals [23]. The transient
expression of His-tagged constructs allows rapid assessment
of expression levels, protein stability and propensity to
crystallize. An initial survey of RPTPμ constructs covered
the entire extracellular region, the MAM–Ig domains and the
MAM domain in isolation. Crystals from preliminary
crystallization screening of the MAM–Ig construct showed
immediate promise for structural studies and screen
conditions were rapidly optimized to yield crystals which
diffracted to 2.7 Å (1 Å = 0.1 nm) resolution [21]. Since
the topology of a MAM-fold was unknown, phasing
was achieved by selenomethionine labelling in the HEK293T expression system and collection of MAD (multiple anomalous dispersion) data at BM14, the UK MAD
beamline at the European Synchrotron Radiation Facility.
The crystals were grown without removal or trimming of the
wild-type complex sugars, and indeed the crystallographic
electron-density maps revealed sugars at three N-linked
glycosylation sites. The MAM domain was revealed to consist
of a β-barrel-type fold commonly referred to as a jelly-roll
fold [21]. This topology has been found in numerous human
proteins, e.g. the cytokine TNF (tumour necrosis factor) [24].
Comparison against the database of known jelly-roll fold
structures scores highest similarity for the RPTPμ MAM
domain and the ephrin-binding domains of the Eph receptor
tyrosine kinases; an intriguing observation given that, like
RPTPμ, the Eph receptors are involved in the development
of the nervous system and the vasculature [25,26]. Within the
MAM–Ig structure, there is a seamless interface between
the β-barrels of the MAM and the Ig domains [21].
The minimal adhesive unit
The aim of determining the MAM–Ig structure was, of course,
not only simply to reveal the architecture, but also to assess
the adhesive functionality of the molecule. In this respect, it
was disappointing to find that the MAM–Ig fragment was a
monomer in solution as assessed using gel filtration, analytical
ultracentrifugation and dynamic light scattering, even at high
protein concentrations. This observation remained true in the
context of the crystal packing, where the lattice showed no extensive intermolecular contact between MAM–Ig molecules
that could represent an adhesive interaction. This prompted
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properties of a series of domain-deletion constructs for the
RPTPμ extracellular region [21]. Gel filtration then indicated
that the MAM, Ig and the first of the FN domains constitute
the minimal fragment capable of mediating a homodimeric
interaction. All constructs containing these, plus subsequent
domains up to the full-length extracellular region, showed
homodimerization. However, the stability of these highaffinity dimers was pH-dependent; dynamic light scattering
measurements indicated that, for pH values below 6.2, dimers
dissociated into monomers. The pH-controlled switch is
reversible: dimers reformed on titration back to pH values
above 6.2. This molecular-level behaviour matches the pHdependency of RPTPμ-mediated cell adhesion observed in
cell-aggregation assays [11,27]. We also took our series of
domain-deletion constructs and tested their adhesive function
in a cellular context by expression in naturally non-adherent
Sf9 insect cells. For this analysis, we used ectodomain
constructs containing a membrane-spanning helix followed
by a GFP (green fluorescent protein) domain replacing
the distal phosphatase cytoplasmic domain. Fluorescence
and confocal microscopy revealed the cellular location and
adhesive activity of the molecules. For transfection of a
construct expressing only the GFP, the cells remained nonadherent as they did for the constructs encoding ectodomains
consisting of merely the MAM domain or the MAM–Ig unit,
or indeed the MAM–Ig–first FN domain. Only constructs
containing the MAM–Ig and two, or more, of the FN domains
were capable of inducing cell aggregation. Thus it was clear
that, to relate the functional role of the extracellular region
to structure, we must focus our crystallographic analyses on
larger fragments, spanning from the RPTPμ N-terminus to
include one or more FN domains, and, ideally, the entire
extracellular region.
Structure of the RPTPμ ectodomain
We returned to our mammalian-cell-based transient expression system; however, given 12 predicted N-linked
glycosylation sites in the full-length RPTPμ extracellular
region, the heterogeneity introduced by the addition of wildtype complex sugars was too great to allow growth of wellordered crystals. We therefore employed a glycosylationdeficient cell line, HEK-293S-GnTI− [28], which lacks
N-acetylglucosaminyltransferase 1 activity, resulting in secreted proteins bearing only simple sugars of the Man5GlcNAc2
type which can be truncated back to a single GlcNAc moiety
on treatment with endoglycosidase H [23]. This deglycosylation treatment, plus maintenance of neutral pH, was essential
for the growth of well-ordered crystals of the functional adhesion molecule. These crystals were capable of diffraction to
3 Å resolution and thus facilitated the structure determination
of the full-length extracellular region of RPTPμ [5]. The
structure revealed an extended rigid conformation. Indeed,
the molecule resembles a textbook schematic diagram showing six domains arranged contiguously as beads on a string
(Figure 1A). The interfaces between each of these domains are
Biochemical Society Annual Symposium No. 75: Structure and Function in Cell Adhesion
Figure 1 Structure and function of the RPTPμ extracellular region
(A) Crystal structure of the RPTPμ ectodomain reveals an extended conformation with domains arranged in a ‘beads on a
string’ fashion. Two molecules form a trans (antiparallel) dimer, held together by multiple interactions over a large contact
surface (1630 Å2 per monomer). Residues from the four N-terminal domains of each molecule (MAM, Ig, FN1 and FN2)
contribute to these interactions [5]. The membrane-proximal FN domain (FN4), although present in the electron-density
map, is not well ordered and therefore is shown here as a rectangle. (B) Structural and functional data suggest that the
RPTPμ ectodomain controls the subcellular localization of the receptor. In the acidic environment of the secretory vesicles, the
molecules are monomeric, but, once they reach the cell surface (and the pH is neutral), they can form adhesive interactions
[11,22]. Such interactions can only happen if the distance between apposing cells is permissive, i.e. at least equal to the
maximal length of the trans dimer. Under such conditions, the ectodomains ‘lock’ RPTPμ molecules at cell contacts (in a
spacer-clamp mechanism) and they accumulate at cell contacts [7,29], in close proximity to their physiological substrates:
members of the cadherin–catenin complex [8,16,36]. The perpendicular orientation of RPTPμ to the cell surface is not
absolute: it is expected that a certain degree of variation will be allowed by the apparently flexible juxtamembrane region
(as also proposed for cadherins [32]). Nevertheless, the free diffusion of RPTPμ is restricted further by the ectodomain size,
since it cannot access narrow intercellular spacings such as the tight junctions [37]. Further interactions between RPTPμ
molecules, as well as between cadherins, probably in cis, have been proposed [19,21,27] (indicated here by arrows and
question marks) but these remain to be confirmed by structural studies.
extensive and apparently very rigid; in addition, there is an
interdomain disulfide bridge between the second and third
FN domains. The residues involved in these interfaces are
highly conserved between members of the type IIb RPTP
family (μ, ρ, κ and λ) and across species (human, mouse, dog,
chicken, frog and fish). This degree of conservation strongly
suggests that the rigid extended ectodomain architecture seen
in the RPTPμ crystal structure has a functional role. The
only exception to the rigidity of the architecture appears to
occur at the fourth FN domain. For this region, the electron
density of the crystal structure is very weak and indistinct,
indicative of inherent flexibility. This may, in part, be the
result of the proteolytic cleavage which occurs in this domain
during the natural processing of the RPTPμ molecule [29].
This cleavage does not result in shedding of the receptor from
the cell surface, but may introduce some flexibility into the
structure of the membrane-proximal FN domain.
In addition to highlighting the interdomain interfaces, the
sequence conservation between RPTP IIb family members
and across species, when mapped onto the RPTPμ structure,
also reveals large patches of highly conserved surface. In the
crystal structure, much of this conserved surface can be seen
to contribute to a trans (i.e. head-to-head) dimer interface.
The extent and nature of this interface bore all the hallmarks of
a physiologically relevant interaction. The interface involves
some 3000 Å2 of buried surface area consisting of a mixture of
interactions involving hydrophobic residues, such as tyrosine
and tryptophan, as well as charge complementarity between
polar, acidic and basic residues. Interestingly, these residues include among their number several histidine residues,
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which could, at least in part, be responsible for the observed
pH-dependency of RPTPμ-mediated adhesion. As the trans
dimer crystal structure appeared to be a very compelling
candidate for the adhesive interaction, we sought to confirm
the hypothesis by mutation of residues which played a
central role in the interface. Six residues were selected for
mutagenesis, of which two were hydrophobic (Trp279 and
Tyr277 ), three were basic (Arg219 , Arg220 and Arg389 ) and one
was a histidine (His167 ). We again used a cell-adhesion assay
based on transfection of normally non-adherent Sf9 cells
with mutant constructs of RPTPμ incorporating cytoplasmic
GFP tags. All of the ectodomain mutants tested lacked the
adhesive activity of the wild-type ectodomain [5].
Does the high level of conservation at the adhesive
interface indicate that interactions can occur between
different members of the RPTP type IIb family? A detailed
inspection of the adhesive interface argues against such
cross-recognition; for example, structural mapping of the
sequence conservation between human RPTPμ and RPTPκ
revealed ten non-conserved residues at the dimer interface
which could contribute to specific homophilic adhesion, i.e.
RPTPμ adhering only to RPTPμ and not to RPTPκ. This
specificity is consistent with the observations of Zondag
et al. [30] for cells expressing either RPTPμ or RPTPκ
constructs and labelled with different fluorescent dyes. The
two cell populations did not intermingle, but rather red
cells adhered to red cells, and green to green; in contrast,
for the control experiment, cells labelled with either red or
green dyes, but all expressing RPTPμ molecules, formed
mottled clumps containing a mixture of red and green cells.
A similar segregation between cells expressing different
type IIb RPTPs can be observed in an anatomical context,
e.g. in multilayered structures such as the cerebellum [14,31].
Functional implications of the adhesive
dimer structure
A combination of structural and functional studies indicates
that RPTPμ, and, by extrapolation, other members of the
type IIB family, are very rigid extended molecules which
show highly specific adhesive properties that generate trans
homodimers. How might these properties relate to the role of
type IIb RPTPs at the adherens junctions? As stated above,
there are multiple lines of evidence to indicate a close functional interplay between RPTPμ and members of the cadherin
family of adhesion molecules, and, indeed, the cadherins
are important inhabitants of the adherens junctions. If we
consider the structural data available for cadherins, the crystallographic analysis of the entire C-cadherin ectodomain by
Boggon et al. [32] revealed a trans dimer capable of spanning a
maximal cell–cell spacing of some 385 Å [the authors suggest
a dimer orientation tilted relative to the cell surface that
would result in a slightly reduced spacing consistent with
that of the adherens junction observed in EM (electron
microscopy) studies]. Our crystal structure of the RPTPμ
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Authors Journal compilation ectodomain trans dimer spans 330 Å, a surprisingly close
match to the reported dimensions of the adherens junctions.
It would appear that the C-cadherin and RPTPμ ectodomain trans dimers are well matched in size to each other
and to the intercellular spacing appropriate for their adhesive
interactions. Why might this be functionally important? If the
cell–cell spacing can in some way influence the concentration
of molecules at particular subcellular locations on the cell
surface, this will provide a mechanism for modulation of
intracellular signalling. One such mechanism, by which
the extracellular cell–cell spacing can have an impact on the
balance between phosphorylation and dephosphorylation
in the underlying cytoplasm, has been proposed for T-cell
signalling. Davis and van der Merwe [33] have developed the
hypothesis that, at localized points of close contact between
the antigen-presenting target cell and the T-cell, size-based
exclusion of the RPTP CD45 may promote kinase-based signalling via the T-cell receptor. The proposed mechanism
is as follows. On engagement of the T-cell receptor with
an MHCI or MHCII antigen-presenting molecule, and the
associated interactions of the co-receptors CD8 and CD4
and adhesion molecules such as CD2 and CD58, the distance
between a T-cell and a target cell is limited to some 150 Å;
the large extracellular region of the phosphatase, estimated at
280 Å by electron microscopy [34], cannot be accommodated
within such close cell–cell spacings and CD45 is squeezed out
from the interface. This depletion of phosphatase activity in
the vicinity of the T-cell receptor would be conducive to Lck
phosphorylation and consequent downstream signalling.
Might the extracellular region of RPTPμ also be acting as a
measure of cell–cell spacing? We postulated that, for RPTPμ,
the properties of the ectodomain served to increase, rather
than decrease, the molecular concentration at a particular
intercellular spacing. At the appropriate spacing for an
adherens junction, the formation of the adhesion trans
dimer could lock the phosphatase activity into position,
i.e. the extracellular region of RPTPμ acts as a spacer clamp
[5].
The spacer-clamp hypothesis
We sought to test our spacer-clamp hypothesis by first
assessing whether RPTPμ accumulates at adhesive interfaces
in our Sf9 cell model system. Immuno-EM analysis of
cryo-sections taken for cell aggregates indeed confirmed
that RPTPμ became concentrated at these interfaces,
consistent with the molecule becoming locked into position
by formation of the trans dimer. Do these normally
non-adherent cells display a distinctive cell–cell spacing defined by the RPTPμ trans dimer? And, if so, does this spacing
become smaller as the length of the RPTPμ ectodomain (and
hence the trans dimer) is shortened by stepwise deletion of
the fourth (membrane-proximal) FN domain and then, in
addition, deletion of the third FN domain (neither domain
is required for cell adhesion)? We used three constructs (the
full-length wild-type ectodomain, and the single- and
the double-FN-domain-deletion mutants) to explore these
Biochemical Society Annual Symposium No. 75: Structure and Function in Cell Adhesion
questions. EM analysis of the resultant cell aggregate
sections, using negative staining, revealed a clear correlation
in cell–cell spacing with length of trans dimer. This behaviour
is fully consistent with our spacer-clamp hypothesis.
The currently available structural and functional data
for RPTPμ can be integrated into a model for adhesionbased regulation of subcellular phosphatase concentrations
(Figure 1B). Before reaching the cell surface, RPTPμ is
transported in secretory vesicles, micro-environments which
are typically at pH 5.5; the acidic pH prevents formation
of trans dimers. On reaching the cell surface, the RPTPμ
is exposed to neutral pH values favourable to formation of
the trans dimer; however, because of the rigid nature of the
molecule, such trans dimers will only form at appropriate
intercellular spacings. At an adherens junction, RPTPμs from
opposing cells can form a trans dimer, fixing the RPTPμ at
this location on the cell surface and, in so doing, locking the
phosphatase activity into position. This local concentration
of phosphatase activity is juxtaposed precisely with the
high local concentration of the cadherins; the dimensions
of the RPTPμ and cadherin cell-adhesion complexes
both match the cell–cell spacing of the adherens junction.
Thus phosphatase activity (RPTPμ) and substrate (the
cadherin–catenin complex) are co-located in the cytoplasm,
and the dephosphorylated state of the cadherin–catenin
complex contributes to the stability of the adherens junction
[19,35].
Questions remain open as to whether there are direct
interactions between RPTPμ and cadherin ectodomains, and
whether ordered arrays (involving both cis and trans adhesive
interactions) form at the adherens junction, either separately
for cadherin and/or RPTPμ or jointly. Is co-localization at
the adherens junctions sufficient or is the precise arrangement
of cadherin and RPTP orchestrated further? These questions
remain to be explored in the future, as does the generality
of the hypothesis that the extracellular regions of molecules
such as the RPTPs are engaged in control of location as a
mechanism to modulate signalling activity within the cell.
We thank Linda Vincent for her expert assistance in preparing
this review. A.R.A. is a U.K. Medical Research Council Career
Development Fellow, C.S. is a Wellcome Trust Career Development
Fellow and E.Y.J. is a Cancer Research UK Principal Research Fellow.
We also thank colleagues who contributed to the structural and
functional characterization of RPTPμ: Kaushik Choudhuri, Veronica
Chang, Weixian Lu, Simon Davis and Anton van der Merwe.
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Received 11 January 2008
doi:10.1042/BST0360167