The EMBO Journal vol.12 no.3 pp.933-941, 1993
Tyrosine 785 is a major determinant of Trk -substrate
Axel Obermeierl, Hartmut Halfter',
Karl-Heinz Wiesmuller2, Gunther Jung2,
Joseph Schlessinger3 and Axel Ulrich1'4
1Department of Molecular Biology, Max-Planck-Institut fiir Biochemie,
Am Klopferspitz 18A, 8033 Martinsried, 2Institut fiir Organische
Chemie, Eberhard-Karls-Universitat Tiibingen, Auf der Morgenstelle
18, 7400 Tiibingen, Germany and 3Department of Pharnacology, New
York University Medical Center, 550 First Avenue, New York, NY
Communicated by A.Ullrich
receptor/Trk with cellular substrates was investigated by
transient co-overexpression in human 293 fibroblasts
using ET-R, a chimeric receptor consisting of the
epidermal growth factor receptor (EGF-R) extraceliular
ligand binding domain and the Trk transmembrane and
intracellular signal-generating sequences. The chimera
fully functional, and associated
phosphorylated phospholipaseC7y(PLC-y), ras GTPase-
activating protein (GAP) and the non-catalytic subunit
dependent manner. Deletion of 15 C-terminal amino
acids, including tyrosine 785 (Y-785) abrogated receptor
and substrate phosphorylation activities. Mutation of
Y-785 to phenylalanine somewhat impaired receptor
phosphorylation activity, which was reflected in reduced
GAP and p85 phosphorylation. In contrast, ET-YF
phosphorylation ofPLCywas significantly reduced, while
the high affinity association potential with this substrate
was abrogated by this point mutation in vitro and in intact
cells. Furthermore, a tyrosine-phosphorylated synthetic
cytoplasmic domain association with PLCy. Thus, the
short C-terminal tail appears to be a crucial structural
of the Trk cytoplasmic domain, and
phosphorylated Y-785 is a major and selective interac-
tion site for PLC-y.
Key words: nerve growth factor/signal transduction/tyrosine
p85, in a ligand-
The missing link between nerve growth factor (NGF) and
cellular signalling mechanisms that lead to the formation of
neurites has been identified by the recent finding that the
proto-oncogene product gpl4Ot* (Trk) (Martin-Zanca et al.,
activity, is a functional receptor for this neurotrophic factor
(Kaplan et al., 1991a,b; Klein et al.,
tyrosine kinases (RTKs) such as epidermal growth factor
receptor (EGF-R), platelet-derived growth factor receptor
a transmembrane receptor with tyrosine kinase
Oxford University Press
(PDGF-R) and macrophage colony stimulating factor
receptor (CSF-lR) are known to function as molecular
switches that transduce extracellular binding to their cognate
ligand into intracellular events, ultimately resulting in DNA
synthesis and cell proliferation or differentiation (reviewed
in Ullrich and Schlessinger, 1990; Cantley et al., 1991).
Ligand binding induces receptor dimerization (Schlessinger,
1988; Ullrich and Schlessinger, 1990) and autophosphory-
lation, followed by association and tyrosine phosphorylation
of a specific subset of cellular protein substrates. Among
these are the RTK substrates phospholipase COy (PLC'y),
GTPase-activating protein (GAP) and the p85 subunit of
phosphatidylinositol 3'-kinase (PI3'-K), which are thought
to be involved in distinct intracellular signal transduction
pathways. Different RTKs appear to utilize distinct sets of
signalling polypeptides to exert their final effects. For
example, PLC&y is a substrate for the tyrosine kinases of
PDGF-R (Meisenhelder et al., 1989; Wahl et al., 1989;
Margolis et al., 1990), fibroblastic growth factor receptors
(FGF-Rs) (Burgess et al., 1990; Mohammadi et al., 1991)
and EGF-R (Margolis et al., 1989; Meisenhelder et al.,
1989; Nishibe et al., 1989), but not for CSF-1R (Downing
et al., 1989) and insulin receptor (Nishibe et al., 1990). In
addition, GAP has been found to be a substrate for EGF-R
and PDGF-R (Kaplan et al., 1990; Kazlauskas et al., 1990),
but not for basic FGF-R (Molloy et al., 1989).
PLC-y, GAP and p85 are thought to bind to phospho-
tyrosine (PY) residues on their respective phosphorylated,
therefore activated, RTKs via their src homology region 2
(SH2) domains (Moran et al., 1990; Cantley et al., 1991;
Koch et al., 1991). Variations in SH2 domains, together with
different amino acid sequences surrounding PY residues, are
likely to be responsible for specific substrate binding to
particular PY residues on distinct receptors (Koch et al.,
1991). For example, Y-992 and Y-1068 on human EGF-R
and Y-766 on chicken FGF-R (Flg) have been identified
as high affinity binding sites for PLC'y SH2 domains
(Mohammadi et al., 1991, 1992; Peters et al., 1992; Rotin
et al., 1992). Similarly, tyrosine residues in the C-tail of
mouse and human ,BPDGF-R appear to be involved in
receptor-PLCy interactions (Ronnstrand et al.,
Seedorf et al., 1992).
Although accumulating evidence points to Trk as the only
component of the high affinity, signal-generating NGF-R
(Klein et al., 1991; Weskamp and Reichardt, 1991; Ibaniiez
et al., 1992; Meakin et al., 1992), it is not yet entirely clear
whether the low affinity NGF-R, p75LNGFR, iS necessary in
addition to Trk to form a high affinity binding site for NGF
and to elicit a full biological response (Hempstead et al.,
1990, 1991; Ragsdale and Woodgett, 1991). To address this
question further and to investigate the significance of
structural domains of Trk signal generation, we utilized a
receptor chimera approach that had been instrumental in
previous studies of RTK function (Riedel et al., 1986;
Lammers et al., 1989; Seedorf et al., 1991). The ET-R
A.Obermeier at al.
Fig. 1. Schematic diagram of receptors. EGF-R extracellular domain
cysteine-rich regions are shown as black boxes; black circles in the
extracellular domain of Trk indicate single cysteine residues. Tyrosine
kinase domains are represented by shaded (EGF-R) and open (Trk)
chimera, consisting of EGF-R extracellular sequences and
Trk transmembrane and cytoplasmic domains, was fully
active in ligand-dependent auto- and substrate phos ho
tion functions, which further confirmed that p75LNGFK
dispensable for NGF signalling through the Trk system. The
demonstrating that PLCy, GAP and p85 are substrates of
the Trk tyrosine kinase. Surprisingly, deletion of the 15
amino acid C-terminal tail rendered the Trk kinase inactive
with respect to autophosphorylation as well as substrate
phosphorylation. Most importantly, our data demonstrate that
the C-terminal-most tyrosine residue of Trk, Y-785, is an
essential part of the PLC-y-binding site.
Expression and autophosphorylation activities of ET-R
To examine the signalling capacity of Trk, we constructed
a chimeric receptor, ET-R, which consists of the EGF-R
extracellular domain fused to the transmembrane and
cytoplasmic sequences of the NGF receptor, Trk (Figure 1).
In addition, three mutations were introduced into the ET-R
background, yielding a kinase-negative point mutant, ET-
KM, a C-terminal truncation mutant, ET-ACT, and a point
mutant, ET-YF, containing a phenylalanine in place of the
C-terminal domain tyrosine residue at position 785 (Figure
Expression plasmids coding for ET-R, ET-KM, ET-ACT
and ET-YF were transiently transfected into a human
fibroblast cell line, 293. Metabolic radiolabelling with
[35S]methionine and immunoprecipitation demonstrated that
all chimeric receptors were synthesized, each appearing as
two bands in SDS-PAGE (Figure 2). The upper bands,
presumably representing the fully processed receptors,
migrated at molecular weights of 142 kDa (ET-R and point
mutants) and 141 kDa (ET-ACT). In each case, the lower
bands migrated at 130 kDa and probably represent high
mannose oligosaccharide-bearing receptors, which fail to exit
the endoplasmic reticulum because they are preferentially
localized inside the cells.
EGF addition to intact transfected cells resulted in a
significant increase in autophosphorylation activity of ET-
Fig.2.Expression andtyrosine phosphorylation of ET-R and mutants.
293 cells transiently expressing ET-R, ET mutants, or parent receptors
(EGF-R andTrk) andmetabolically radiolabelled with [35S]methionine
were stimulated with EGF(EGF-R, ET-R and mutants; +) or NGF
(Trk; +), or left unstimulated (-), lysed and thenimmunoprecipitated
with mAb 108.1 (caEGF-R) (lanes 1-10) or ATC (caTrk) (lanes 11
and 12). Samples weresubjected to 7.5% SDS-PAGE and
electrophoretically transferred to nitrocellulose. Expression levels were
monitoredby autoradiography (upper panel). Phosphotyrosine contents
of the same receptor proteins were analyzed by immunoblotting with
mAb 5E2 (aPY) (lower panel).
R and ET-YF(Figure 2, lowerpanel, lanes 1, 2, 7 and8).
Thephosphorylation activity ofET-YF was somewhat lower
than that ofET-R, indicating that the C-terminaltyrosine
might represent an autophosphorylation site. Asexpected,
ET-KM did notdisplay any kinase activity, yet surprisingly,
phosphorylation of ET-ACT could also not be detected.
Receptor phosphorylation is thought to occur by inter-
molecular transphosphorylation between dimerized
oligomerized receptors in response to ligand stimulation
Transphosphorylation studies employing EGF-R-insulin
receptor chimerae (Ballotti et al., 1989; Lammers etal.,
1990)or an EGF-R-c-kit chimera (Herbst et al., 1991) have
suggestedthat bothhomologous extracellular orcytoplasmic
domainsmay be sufficient to mediate receptor dimerization
demonstrated that transphosphorylation can occur even
between RTKs as distantly related as Trk and EGF-R. As
shown inFigure 3 (lanes 7-10), the kinase-deficient EGF-
R-KA mutant (Honegger et al., 1987) wasphosphorylated
ontyrosines whencoexpressed with the Trk tyrosine kinase
interestingly, transphosphorylation of kinase-negative ET-
KM by EGF-R was not detectable. Thus, Trk cytoplasmic
sequences do not appear to be a substrate for the EGF-R
tyrosine kinase in intact cells, while the reverse ispossible.
Tyrosine phosphorylation of cellular proteins by ET-R
Distinct substrate specificities of different RTKs result in
receptor- and cell type-specific pleiotropic responses (for
Trk- substrate interaction
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Received on October 2, 1992; revised on December 2, 1992