2. General Introduction of Cytokine Receptors
Cytokine receptors such as the EpoR, prolactin, and thrombopoietin
receptors function as ligand-induced or ligand-stabilized 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. A four-residue hinge region links subdomains 1 and 2.
Many of these structural characteristics can be seen in figure 4.
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3. 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.
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4. 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.
5. 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
6. 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.
7. Endocrinology 143(1): 2-10.
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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. This
brings up a very significant difference. RTKs possess intrinsic tyrosine kinase
8. 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.