Hibret Adissu
Huixia Zhang
Jiae Paik
Israel Huff
RTK and Cytokine Receptors
Brief Overview of Structure and Function
Every aspect of cellular function in a multicellular organism is
dependent on signalling molecules.
In order to accomplish the various
functions, cells sense and react to environmental changes or chemical
signals via signal receptors.
Some chemical signals like the steroid
hormones are membrane permeable and their receptors are located in the
cytoplasm.
In contrast, chemical signals such as growth hormones are
membrane impermeable and are recognized by receptors on the plasma
membrane.
Receptor tyrosine kinases (RTKs) and cytokines are such
receptors and are the focus of this article.
These extracellular
receptors have a similar overall architecture with regard to the
extracellular domain that binds ligands, a single transmembrane helix,
and a cytoplasmic signal-transducing domain.
Based on the presence or
absence of catalytic domains, these receptors can be classified as
receptors with intrinsic enzymatic activity and those without it
(Schenk
and Snaar-Jagalska, 1999).
RTKs have intrinsic enzymatic activity through a cytoplasmic
tyrosine kinase domain that catalyzes phosphorylation of protein
substrates including the receptors themselves (Hubbard, 1999).
Most
RTK ligands are soluble polypeptides including insulin, epidermal
growth factor (EGF), and platelet-derived growth factor (PDGF).
RTKs
are involved in processes such as fertilization, proliferation, cell
migration, and apoptosis.
They also play key roles in various
pathological processes including cancer and atherosclerosis (Östman and
Böhmer, 2001).
Binding of a ligand to an RTK receptor induces receptor
dimerization, activation of intrinsic tyrosine kinase activity, and
transphosphorylation of the dimerized monomers.
Phosphorylation
activates the catalytic domain of the receptor and allows access of
substrates and ATP to the catalytic domain.
In addition,
phosphotyrosine residues on the non-catalytic domain of the receptor
create docking sites for adapter proteins such as Gab-1, Sch, and Grb-2
that possess the Src homology (SH) domain.
For instance, SH2 domain-
mediated recruitment of the Grb-2 (Growth factor receptor bound 2) associated Ras guanine nucleotide exchange factor, mSos, allows the
formation of a complex that activates small G proteins such as Ras.
Likewise, many signaling molecules such as phosphatidylinositol 3kinase (PI3K) and Phospholipase C utilize the phosphotyrosine residues
as a docking site for assembly of signaling complexes (Nystrom and
Quon, 1999).
Docking of critical signaling molecules facilitates the
recruitment of other signaling molecules to the plasma membrane to
initiate signaling cascades leading to the activation of nuclear
transcription factors.
Unlike the RTKs, the cytokine receptors lack intrinsic catalytic
domains.
Instead, the Janus Kinases (JAKs), which are non-covalently
associated with the receptor, couple tyrosine phosphorylation with
ligand binding to the receptor (Gadina et al., 2001).
JAK1, JAK2, JAK3, and TyK2.
The JAKs include
The binding of a cytokine to cytokine
receptor causes receptor dimerization and the activation of JAKs.
The
receptor is then phosphorylated at specific tyrosine residues and
serves as a docking site, via the SH2 domain, for transcription factors
termed STATs (Signal Transducer and Activators of Transcription).
This
is followed by phosphorylation of STATs by the JAKs, dimerization of
the STATs, and translocation of them to the nucleus where they can
carry out gene activation (Schuai, 1999; Frank, 2002).
Various
cytokines including the interleukins, erythropoetin (EPO), and growth
hormone (GH) utilize this pathway (Shuai, 1999).
Cytokines play critical roles in various biological processes
including the immune response, hematopoiesis, and apoptosis.
also involved in numerous pathological conditions.
They are
For instance, Human
Severe Combined Immunodeficiency (SCID) can result from mutations in
the cytokine receptors JAK-3 and Il-7 (Buckley et al., 2001).
Due to their diverse and critical biological functions, the RTKs
and the cytokine receptor signaling systems have been the targets of
extensive research.
step in signaling.
Ligand-receptor complex formation is the primary
Consequently, there is considerable interest in the
structural analysis of ligand-receptor complexes.
Such undertakings
have been instrumental to the discovery of novel therapeutic molecules.
For example, EGF receptor tyrosine kinase inhibitors like ZD1839
(Arteaga and Johnson, 2001) have been developed for cancer treatment.
Likewise, EMP-1, a peptide agonist of the erythropoetin receptor
(Wrington et al., 1996) and small non-peptidyl molecule with
thrombopoetin activity (Duffy et al., 2001) has been developed.
Receptor Tyrosine Kinases
Inactive and Active States of RTKs
RTKs are membrane receptors with three domains: a glycosylated Nterminal extracellular domain that binds polypeptide ligands, a single
helix that traverses the membrane, and a C-terminal, cytoplasmic domain
that has catalytic activity and sites for interaction between monomers
of the dimer.
There is a great deal of variety in the extracellular
domains of of RTK family members as demonstrated in figure 1.
Furthermore, only a small part of the RTK extracellular domain is
involved in ligand binding (Hubbard et al, 2000).
For example, for the
VEGF receptors, two of the 7 Ig-like domains D2 and D3 are responsible
for ligand binding.
For the NGF receptors, NGF ligand binding is
carried out by the 2 Ig-like domains, which follow 2 cysteine-rich
domains interrupted by a leucine rich domain.
For the EGF receptor
family, the cysteine-rich domain functions as the ligand-binding site.
Compared to the diversity in the extracellular domains, the cytoplasmic
domains are more uniform across the RTK family.
They all have a
tyrosine-rich domain followed by the C-terminal domain.
Autophosphorylation of tyrosines in the tyrosine-rich domain stimulates
receptor catalytic activity and creates docking sites for downstream
signalling proteins.
Though there is little diversity in the
cytoplasmic domains, some receptors such as the PDGF (Pallet Derived
Growth Factor) receptor contain a large insertion in the tyrosine
kinase domain.
This insertion can be involved in regulation of
receptor activity.
_______________________________________________________________________
Figure 1.
Domain
organization for a
variety of RTKs.
The
extracellular domain
of the receptors is
on the top and the
cytoplasmic domain is
on the bottom.
Progress in Biophysics and Molecular Biology 71: 343-358.
Most RTKs exist as monomers in the absence of ligand.
however, two exceptions in the family.
There are,
The Met (hepatocyte growth
factor receptor) and the insulin receptor subfamilies (Hubbard et al,
2000).
Met has a short alpha-helix disulfide-linked to a beta-strand
that spans the membrane.
In the case of insulin receptor, there are
two extracellular alpha-helices linked to two membrane-spanning betastrands via disulfide bonds.
Therefore, unlike the other RTKs, the
receptors of these subfamilies exist as dimers in absence of bound
ligand.
The mechanism by which most RTKs transduce signals can be
described as follows (Kroiher M et al, 2001): ligand binding induces
dimerization of receptor monomers, dimerized monomers
transphosphorylate one another, downstream proteins bind to receptor
phosphotyroinses, and phosphorylation of these docked proteins and
other substrate proteins takes place.
The possible mechanism for
signal transduction by various RTKs is illustrated by Figure 2.
_______________________________________________________________________
Figure 2.
Activation and
phosphorylation by RTKs.
A) homodimeric receptor and
ligand.
B) heterodimeric.
Bioessays 23: 69-76.
Dimerization
Ligands bind to substrate binding domains in the extracellular
regions of RTKs.
This stabilizes dimerization of monomeric receptors
via noncovalent interactions or, in the case of heterotetrameric
receptors like insulin receptors, induces a change in their quaternary
structures to stabilize the tetramer (Hubbard, 1999).
state, some RTKs bind one ligand while others bind two.
In the dimeric
For example,
one PDGF ligand binds to two receptor dimers via two equivalent binding
sites acting in unison (the PDGF:PDGFR dimer ratio is 1:2).
EGF forms
a complex of two ligands and two receptor dimers (the EGF:EGFR dimer
ratio is 2:2) (Lemman et al, 1997a).
FGF interacts with its receptor,
FGFR, in a simple 1:1 ratio(Hubbard, 1999).
It should now be clear
that several different configurations of ligand-receptor complexes
exist in the RTK family.
It is, however, the 1:1 ratio that is most
common.
It is now accepted that ligand binding stabilizes the association
of the extracellular domains of the monomers of RTK dimers.
There are,
however, two different views regarding the stability of the association
of the cytoplasmic domains (Hubbard, 1999).
In one proposed model, the
cytoplasmic domains associate only transiently, acting simply as
substrate and enzyme to one another.
In the other model, they form a
stable dimer much like those formed by the extracellular domains.
Schlessinger proposed that active dimers can exist in the absence of
bound ligand since autophosphorylation of RTKs can be enhanced by
inhibitors of protein tyrosine phosphotases even when no ligand is
present (Schlessinger, 2000).
In this case, receptors would be in an
equilibrium between monomeric and dimeric states.
The presence or
absence of ligand would shift the equilibrium to the dimeric or
monomeric states, respectively.
This is demonstrated in figure 3.
At
any rate, ligand binding stabilizes the active dimer form of receptors
or induces a structural rearrangement in heterotetrameric receptors.
Thus, it facilitates tyrosine-transphosphorylation between the
cytoplasmic domains of the two monomers (Heldin, 1995).
_______________________________________________________________________
Figure 3.
Receptor state
equilibria vary
with
phosphorylation
state and
ligand
presence.
Cell 103: 211225.
_______________________________________________________________________
Autophosphorylation
Phosphorylation of the tyrosines in the active loop (A loop) of
the tyrosine kinase (TK) domain stimulates the intrinsic catalytic
activity of the TK domain.
The phosphorylated tyrosines in the A loop
and the C-terminus function as binding sites for proteins that
recognize the phosphorylated amino acids using motifs like the SH2
domain and the phosphotyrosine-binding (PTB) domain (Hubbard et al,
2000).
All known RTKs contain one to three tyrosines in the A loop
that are involved in receptor function.
For instance, one of the
tyrosines in the insulin receptor TK domain binds to the active site,
preventing the peptide substrate from accessing it (Till et al, 2001).
Phosphrylation of this tyrosine can reduce its affinity for the active
site, freeing the site for substrate binding.
Autophosphorylation of the A loop brings about a dramatic
structural change of the loop (Hubbard, 1999).
Prior to
autophosphorylation, the two lobes at the N- and C-terminuses of the
loop are kept away from each other by residues located at the beginning
of the loop.
Autophosphorylation of A loop tyrosines causes the N-
terminal lobe to rotate towards the C-terminal lobe, taking the
residues of the A loop responsible for Mg-ATP binding into the right
orientation to facilitate binding.
Therefore, autophosphorylation of
the A loop allows Mg-ATP and peptide substrates access to their binding
sites and repositions the residues involved in substrate binding and
catalysis.
Mg-ATP binds at the beginning of the A loop via the Asp of
the DGF motif, while the peptide binding site is at the end of the
loop.
Though the mechanism of activation just discussed is generally
carried out by RTKs, some RTKs require more stringent conditions for
activation.
One such exception is activation of the EGF (epidermal
growth factor) receptor.
Simply linking the two receptor monomers is
not sufficient for EGFR activation, so some other conditions must be
met.
The relative orientation of the two monomers in the dimeric
structure, especially rotation of the flexible cytoplasmic domains
caused by rotation of the transmembrane domain, may play an important
role in activation of the intrinsic catalytic activity of EGFR (Moriki
T et al, 2001).
Cytokines
General Structure of Cytokine Receptors
Cytokine receptors such as the EpoR, prolactin, and
thrombopoietin receptors function as ligand-induced or ligandstabilized homodimers.
They interact with the Janus (JAK) family of
cytoplasmic tyrosine kinases to participate in signal transduction.
Erythropoietin and other cytokine receptors are activated through
hormone–induced receptor dimerization and autophosphorylation of JAK
kinases that are associated with the cytoplasmic domains of the
receptors.
In the case of cytokine receptors such as Epo-like
receptors and growth hormone receptors, the receptor consists of an
extracellular ligand-binding domain, a short single-pass transmembrane
domain, and a cytoplasmic domain that lacks tyrosine kinase activty. In
the extracellular domain, there are about 200 to 250 amino acid
residues comprising two subdomains (1 and 2), each predicted to consist
of seven beta-strands and to be structurally related to fibronectin
type III (FN III) domains (Barzan, J.F., 1990). The amino-terminal
FNIII-like subdomain contains a pair of spatially conserved cysteine
bridges, while the carboxyl-terminal FNIII-like subdomain contains a
conserved β-stand F and a highly conserved WSXWS motif.
hinge region links subdomains 1 and 2.
A four-residue
Many of these structural
characteristics can be seen in figure 4.
_______________________________________________________________________
Figure 4.
Dimerized cytokine
receptor with ligand bound.
Endocrinology 143(1): 2-10.
The single transmembrane domain and part of the juxtamembrane
domain are proposed to be alpha-helical.
Three hydrophobic amino
acids, L253, I257, and W258, found in the juxtamembrane domain are crucial
for receptor signaling.
These three hydrophobic residues are predicted
to form a hydrophobic patch on the alpha-helix (Constantinescu et al.,
2001).
This segment is also specifically required to switch on JAK
activation.
It has been proposed that the juxtamembrane domain is
important both in activation of JAK and in positioning of the
cytoplasmic domain of Epo-like receptors in the conformation to be an
acceptable JAK substrate (Constantinescu et al., 2001).
The importance
of the juxtamembrane domain helix and the three hydrophobic residues
mentioned are schematically represented in figure 5.
_______________________________________________________________________
Figure 5.
Importance of L253
positioning in the
juxtamembrane domain for JAK
activation.
A) wild-type,
functional.
B) L253A
knockout, disabled.
C)
juxtamenbrane domain
lengthened 1 aa so L253 is
not positioned in the
hydrophobic patch, disabled.
D) lengthened 3 aa so L253 is
again positioned in the
hydrophobic patch,
functional.
Mol. Cell Biol. 7: 377-385.
The cytoplasmic domains of the receptors have JAK binding domains
and several tyrosines involved in recruiting other signaling proteins
such as STAT by providing docking sites.
When the ligand binds to the
extracellular domain, it triggers transphosphorylation of the JAKs
bound to each of the receptor monomer cytoplasmic domains activating
the tyrosine kinase activity of the JAKs.
The JAKs induce
phosphorylation of tyrosines in the cytoplasmic domains in each of the
receptor monomers.
This subsequently leads to docking of signalling
proteins such as STAT, PI-3’ kinase, the protein tyrosine phosphatases
SHP1 and SHP2, and Shc to the tyrosine residues.
Several of the
tyrosines of these bound proteins, in turn, become phosphorylated by
the JAKs.
Conformational Changes in Cytokine Receptors Induced by Ligand Binding
The erythropoietin receptor (EPOR) is activated by ligand-induced
homodimerization.
One ligand binds to the heterodimerized
extracellular domains of the receptor so that it has a 1:2
ligand:receptor stoichiometry.
An interesting feature of this ligand–
receptor interaction is that the ligand has no axis of symmetry.
Two
distinct sites, site 1 and site 2, in the ligand molecule each have
different affinities for the receptor.
Site 1 has a higher binding
affinity to the receptor than does site 2.
However, the ligand
molecules were shown to engage the two extracellular domains of the
receptor monomers at similar contact points on each dimerized receptor
(Stuart J. Frank, 2002).
Cytokine receptors exist as preformed dimers.
Livnah et al.
reported that the crystal structure of unliganded EpoR is a dimer, but
with a dramatically different arrangement of the two subunits from the
ligand-bound EpoR.
Ligand binding to the extracellular domain of two
cytokine receptors induces formation of a receptor dimer of very
specific conformation.
Unliganded receptor dimers exist in a
conformation that prevents activation of JAK, but then undergo a
ligand-induced conformational change that allows JAK to be activated
(Ingrid Remy et al., 1999).
The unliganded receptor is in an open-
scissors-like configuration with the dimerization interface consisting
of self-association of the two ligand-binding sites on the
extracellular binding proteins (EBPs).
In this case, the C-terminal
ends of the subdomain 2 regions of the EBPs are quite far apart (over
70 Å).
In the ligand-bound EBP structures, however, these C-terminal
regions are much closer (30 Å for the Epo-engaged EpoR) as can be seen
in figure 6.
Therefore the preformed dimer, which is in an inactive
state, by keeping the cytoplasmic domains apart brings the
extracellular and cytoplasmic domains into proximity and allows
signalling upon ligand binding.
After reorganization of the EBPs
induced by ligand binding, this specific conformation is transmitted
through the two transmembrane alpha-helices to the two receptor
juxtamembrane domains.
Residues in the first eleven amino acids of
this juxtamembrane domain appear to have an alpha-helical orientation
that is functionally continuous with that of the transmembrane domain.
The segment of JAK is bound, at least in part, to specific amino acids
in this eleven amino acid juxtamembrane domain.
In this case, the
ligand-triggered, receptor-reorganized dimerization brings the two
bound JAK proteins together in such a way that they can phosphorylate
and activate one other.
The activated JAK can then phosphorylate
multiple tyrosines in the two receptor cytoplasmic domains leading to
the phosphorylation of other signaling proteins involved in signal
transduction.
Figure 6.
Large-
scale structural
changes in the
cytoplasmic domains
of cytokine
receptors upon
ligand binding.
Endocrinology
143(1): 2-10.
_______________________________________________________________________
RTK Versus Cytokine Receptors
Both RTK and cytokine receptors have been discussed in
substantial detail in this article so far.
Next, a comparison of the
similarities and differences between the two receptor classes will be
undertaken.
Though the particulars of various receptors in each class
have been mentioned, this comparison will only consider those
characteristics more and less common to each receptor class.
RTKs and cytokines are cell surface signal receptors that share
numerous features in common.
However, they also possess many unique
features that differentiate between the two receptor classes.
The
monomeric units of each receptor class follow a similar three domain
layout: a variable glycosylated extracellular N-terminus for ligand
binding, a single transmembrane alpha-helix for membrane anchoring, and
a conserved cytoplasmic C-terminus that takes part in signaling (though
the C-terminal domains are different between the classes, they are
somewhat conserved within them).
Both RTKs and cytokines exist as
dimers in the active state, yet only cytokines are dimers in the
inactive state.
RTKs exist as monomers when in the inactive state and
dimerize only upon ligand binding.
In cytokines, ligand binding
changes the conformation of the complex of dimer and noncovalently
bound JAKs to activate the JAKs.
difference.
This brings up a very significant
RTKs possess intrinsic tyrosine kinase activity, whereas
cytokines depend on the tyrosine kinase activity of JAKs to serve the
same role.
In either case, transphosphorylation between the monomers
(intrinsic or JAK-mediated) activates the ability of the complex to
carry out tyrosine phosphorylation of other signaling proteins.
Moreover, some of the phosphotyrosine residues generated by
transphosphorylation play a part in forming docking sites for
recruiting and binding these signaling proteins to the complex.
Once
the signaling proteins are phosphorylated, both receptors release them
into the cytoplasm where they can carry out their roles in signaling
pathways.
There is some variability within each class of receptor, but
most receptors in each class share the features just discussed.
Since
the two different classes share many features in common, it is evident
that RTKs and cytokines are functionally related cell surface
receptors.
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