Chemistry Reference
In-Depth Information
stimulated and several tyrosine residues of RTK in the cytoplasmic domain become
phosphorylated. This tyrosine phosphorylation is critical for the signal transduction
of RTKs as the phosphotyrosine residues provide the scaffolds for cytoplasmic
proteins to signal downstream reactions.
Interactions between RTK molecules are indispensable for the transduction of
signals across the plasma membrane. Having only one membrane-spanning do-
main, which is thought to form an
-helix, conformational changes of the RTK
extracellular domain induced by ligand binding should have no in
a
uence on the
structure of the cytoplasmic kinase domain in single molecules. In addition, it is
usually thought that an RTK molecule cannot phosphorylate itself. Actually, ligand-
bound RTKs form homodimers in speci
c structures which are known as signaling
dimmers. Signaling dimer formation changes the steric relationship between two
cytoplasmic domains in the dimers to induce mutual phosphorlyation. In some
cases, such as nerve growth factor (NGF), the ligand is a homodimer which crosslinks
two receptor molecules to form a signaling dimer. In other cases, for example
epidermal growth factor (EGF), two ligands contained in a signaling dimer do not
interact with each other but stimulate an allosteric conformational change in the
receptor molecules to induce formation of the signaling dimer [5].
Two types of RTKs are described in this section: EGF receptor (EGFR) and TrkA
nerve growth factor receptor. Activation of EGFR is responsible for proliferation,
morphological changes, chemotacticmovements and carcinogenesis in various types
of cells. Signals from NGF induce differentiation, chemotactic movements and
survival of nerve cells. NGF recognizes two types of membrane receptors, TrkA and
p75. Only TrkA belongs to the RTK superfamily.
5.3
Association between EGF and EGFR and Formation of the Signaling Dimers of EGFR
Association between EGFand EGFR and formation of the EGFR signaling dimers are
the initial crucial steps in EGF signal transduction. Extensive studies have been
carried out with regard to the association between EGF and EGFR showing that cells
have only one type of receptor for EGF (EGFR, also called ErbB1) but, on the surface
of living cells, there are multiple populations of binding sites which vary in their
af
nity and association and dissociation rates with EGF. On the other hand,
clustering and predimerization have been suggested for at least part of the EGFR
population on the cell surface. However, the relationship between such functional
and structural information is largely unknown [6, 7].
We used EGF conjugated with tetramethylrhodamine (Rh-EGF) at the amino
terminus to trace association processes between EGF and EGFR on living HeLa
cells [8]. Modi
cations at the amino terminus do not inhibit the biological activities of
EGF. HeLa cells were cultured on coverslips and Rh-EGF was applied under a
uorescent microscope. In order to observe the cells apical surface, oblique angle
illumination
fluorescence microscopy was used. The numbers and intensities of
individual
fluorescent spots of Rh-EGF that appeared on the cell surface were
