Biology Reference
In-Depth Information
EPOR activation
EPOR dimerization
Dimerization has been shown to be crucial for EPOR activation. For example,
bivalent, but not monovalent, monoclonal antibodies directed to the extracellu-
lar domain of the EPOR, as well as small noncovalently dimerized peptides, can
activate EPOR [12, 13]. An R129C point mutation in the extracellular domain
renders the receptor constitutively active as a result of the formation of an inter-
molecular disulfide bond that connects two receptor polypeptides [14, 15].
Based on the crystal structure of the growth hormone receptor (GHR) extracel-
lular region and comparison between EPOR and GHR amino acid sequence,
Arg129 maps to a region near the dimer interface. Two other mutations in this
region, E132C and E133C, also constitutively activate the receptor and form
disulfide-linked dimers [16]. Fusion proteins of the extracellular domain of c-kit
or epidermal growth factor (EGF) receptor to EPOR cytoplasmic domain can be
activated by stem cell factor (SCF) or EGF, respectively, which are known to
dimerize their corresponding receptors [17]. Cytoplasmic truncation mutants of
EPOR, which are inactive in cell proliferation assays when expressed on their
own, dominantly inhibit EPO-dependent proliferation and differentiation when
co-expressed with wild-type receptor [16, 18, 19]. In 1998, the crystal structure
of a complex between EPO and the extracellular domain of EPOR was obtained
[20]. This structure shows a 1:2 EPO/EPOR stoichiometry, and leads to a model
that after EPO binding, two monomeric receptors are brought together into a
dimer and activate the receptor. (See Chapter 3 for further information.)
Recent lines of evidence however, reveal that EPOR exists as a preformed
dimer on the cell surface before ligand binding. The crystal structure of the sol-
uble extracellular domain of human EPOR in its unliganded state adopts a
dimeric configuration, although with a geometry different from that of the
EPO-bound receptor [21]. Results of antibody-mediated immunofluorescence
copatching (oligomerizing) of epitope-tagged receptors at the surface further
demonstrated that unliganded EPOR forms dimers or higher-order oligomers
on living cells [22]. Consistently, when two complementary dihydrofolate
reductase (DHFR) fragments were fused to the cytoplasmic domain as a fusion
protein through a long linker (30 amino acids, 120 Å), complementation
between the DHFR sub-units ocurred between unliganded receptors [23].
Specific homophilic interactions between the transmembrane domains have
been shown to play important roles in the oligomerization of EPOR. Kubatzky
et al. showed that the expression of the transmembrane domains of EPOR in a
chimeric bacterial protein triggers transmembrane-mediated dimerization and
the activation of a reporter gene [23, 24]. Abrogation of dimerization by muta-
genesis in EPOR transmembrane domain also impaired EPOR signaling in
mammalian cells. The researcher concluded that interactions between trans-
membrane domains may drive EPOR signaling. Constantinescu et al. showed
that oligomerization of EPOR is dependent on its transmembrane domain.
