42 THE ANATOMICAL RECORD (NEW ANAT.

TUTORIAL

Receptor Crosstalk: Communication Through

Cell Signaling Pathways
STEVEN M. HILL

The response of cells to extracellular stimuli is mediated in part by a number of intracellular signal transduction
pathways. The frequent lack of a one-to-one correlation between receptor activation and intracellular responses, such
as predictable nuclear transcription events, is perplexing. This lack of correlation, however, suggests that various
signaling pathways intersect and crosstalk to modify and influence the biological outcome of a specific extracellular
signal. In this review, the basic pathways and aspects of signal transduction are laid out, and known sites of crosstalk
are discussed. A clearer understanding of receptor and cell signaling pathways and levels of crosstalk should provide
insight into the paradoxes that underlie both imprecision and predictability in signal transduction. Anat. Rec. (New
Anat.) 253:42–48, 1998. r 1998 Wiley-Liss, Inc.

KEY WORDS: signal transduction; receptor; crosstalk

The actions of hormones, growth fac-
tors, and cytokines are mediated
through receptors or binding proteins
that, upon binding their appropriate
ligand, activate signaling mechanisms
that lead to an appropriate biological
response. Thus, all receptor molecules
are transducers of extracellular or in-
tracellular signals. Four categories of
signal transducers account for most of
the endocrine, growth factor, and cyto-
kine actions. These are represented by
trimeric G-protein–coupled receptors
(GPCRs), receptor-tyrosine kinases
(RTKs), cytokine receptor-activated ki-
nases, and members of the steroid/
thyroid hormone receptor superfam-
ily. Over the past several decades, the
field of endocrinology has focused on
the interaction of ligands with their
specific receptors and the identifica-
tion of the specific linear signaling
events associated with those receptors
that lead to distinct biological re-
sponses, often requiring tissue-spe-
cific gene expression. However, a cen-
tral biological problem has developed
Dr. Hill is with the Department of
Anatomy at the Tulane Cancer Center of
the Tulane University School of Medi-
cine in New Orleans, LA.
Grant sponsor: NIH; Grant number:
R01 CA74035–01.
as a result of our increased knowledge
of signal transduction mechanisms, in
particular the discovery that frequently
there is not a direct linear relationship
between the binding of a ligand to its
receptor and the activation of an exclu-
sive signaling pathway.
Phosphorylation of cellular proteins
plays an important role in the control
of cell growth and differentiation in-
duced by growth factors and onco-
genes. Numerous serine/threonine ki-
nases have been identified that may
catalyze these phosphorylation reac-
tions. One of the most promising can-
didates is the family of enzymes known
as mitogen activated protein kinase
(MAP kinase) or extracellular signal
regulated kinase (ERK).1,2 Activation
of MAP kinase is a common event in
many signal-transduction pathways.
The cascade(s) leading to MAP kinase
activation is highly conserved and has
been found in numerous organisms
from yeast to mammals. The activity
of MAP kinase is acutely stimulated
by a plethora of mitogenic stimuli,
including growth factors with tyrosine
kinase receptors, cytokines, T-cell anti-
gens, hormones that bind to G-protein–
coupled receptors, and phorbol esters,
suggesting that this pathway may rep-
resent a site of integration for com-
mon signaling mechanisms in cell
regulation.3 This review describes
these receptors and how they are
thought to interact with each other
and the MAP kinase system to affect
signal transduction.
G-PROTEIN–COUPLED
RECEPTORS
Hormones such as angiotensin, epi-
nephrine, glucagon, melatonin, vaso-
pressin, and pituitary glycoprotein hor-
mones bind to unique members of the
hepathhelical transmembrane recep-
tor superfamily of G-protein-coupled
receptors (GPCRs), which are coupled
to their effectors by heterotrimeric
GTP-binding proteins (G-proteins). G-
proteins are comprised of three sub-
units: a, b, and g (Fig. 1, purple).
Binding of a hormone to its GPCR
alters the conformation of the recep-
tor, enabling it to associate with an
inactive G-protein. The formation of
this complex promotes the dissocia-
tion of the a subunit from the b and g
subunits. Accompanying hormone-
receptor binding and the disassembly
of the trimer is an exchange of GDP
for GTP on the a subunit. This enzy-
matic process, called guanidine nucle-
otide exchange, is a crucial biochemi-
cal process. In fact, toxins such as the
cholera toxin inhibit this activity of a,
resulting in serious acute disease.
Primary effector activities of the G-
TUTORIAL THE ANATOMICAL RECORD (NEW ANAT.) 43

Figure 1. Schematic representation of possible growth factor, hormone, and cytokine signaling pathways to and from the nucleus. The primary
growth factor pathway and RTK pathway is shown in orange, while the stress-activated pathway is highlighted in yellow. The cytokine
receptor–activated pathway is denoted in white, while the GPCR/PKC pathway is highlighted in blue. The different levels of feedback from the
expression of gene products are highlighted in green. For details and abbreviations, see the text and Table 1.

protein are ascribable to the GTP-
bound a subunit and various isoforms
of the a, b, and g subunits which have
diverse effector profiles. There are at
least five b-subunit isoforms and 11
g-subunit isoforms, but the a subunit
is the most diverse, with 23 different
isoforms recognized so far.4 The multi-
plicity of a, b, and g subunits allows
the formation of many heterotrimeric
species which may confer increased
specificity or flexibility of signaling to
different GPCRs. For example, Gs iso-
forms stimulate adenylate cyclase
(which converts ATP to cyclic-AMP
(cAMP), an important second messen-
ger) and increase calcium (Ca21) chan-
nel activity (which regulates Ca21, an-
other second messenger in signal
transduction). Conversely, Gi isoforms
act as inhibitors of adenylate cyclase.
Gq isoforms stimulate phospholipase
Cb, leading to inositol phospholipid
turnover (and affecting yet a third
second messenger). GPCRs can also
alter gene transcription, as will be
discussed later, via interaction with
transcription factors such as the cAMP
response element binding protein
(CREB). CREB is activated by phop-
sphorylation, which can be mediated
by either cAMP-dependent protein ki-
nases or Ca21/calmodulin-dependent
protein kinases5 (Fig. 1, blue).
RECEPTOR TYROSINE KINASES
(RTKs)
Hormones associated with cell prolif-
eration—including fibroblast growth
factor (FGF), epidermal growth factor
(EGF), platelet-derived growth factor,
insulin-like growth factors (IGFs), and
insulin—utilize receptors belonging to
the RTK superfamily (Fig. 1, orange).
In addition, neurotrophins bind to a
related family of RTKs designated Trks.
RTKs typically consist of an extracellu-
lar (receptor) domain and an intracel-
lular (protein tyrosine kinase) domain
joined by a membrane-spanning re-
gion. Upon binding of hormone extra-
cellularly, RTKs dimerize, and the ki-
nase domains phosphorylate themselves
at tyrosine residues (autophosphoryla-
tion). This autophosphorylation creates
intracellular docking sites which recruit
various effector proteins present in the
cytosol. Some of these effectors, such as
phospholipase Cg and c-Src, have intrin-
sic catalytic activity. Others, exemplified
by the growth factor receptor binding
protein 2 (Grb2), have no intrinsic cata-
lytic activity but function as adapter mol-
ecules and recruit other proteins to the
complex. Grb2 contains both Src-homol-
44 THE ANATOMICAL RECORD (NEW ANAT.) TUTORIAL

ogy 2 (SH2) and Src-homology 3 (SH3) the sequential nature of the SAPK
TABLE 1. List of Abbreviations for
domains, amino acid sequences that act pathways, the activation of the rel-
Components of the Various Signal
as binding sites for specific receptors evant MKK/MEK/SAPK kinase re-
and/or cytosolic molecules. For example, Transduction Pathways quires its phosphorylation by an up-
through the SH2 domain, the Grb2 mol- stream kinase analogous to the Raf
Abbreviation Description
ecule interacts with the autophosphory- kinase isoforms that participate in the
lated RTK, while via its SH3 domain AC Adenylate cyclase classical MAP kinase pathway as MKK/
Grb2 can associate with the proline-rich AP-1 Activator protein 1 MEKK/SAPK activators.
region of the Sos molecule, a guanidine CR Cytokine receptors
nucleotide exchange protein. CRE cAMP response ele-
The most important signaling mol- ment
CREB cAMP response ele-
CYTOKINE RECEPTOR (CR)
ecules recruited to the RTK signaling SUPERFAMILY
ment binding pro-
complex are the monomeric GTP-bind-
tein Growth hormone, prolactin, erythro-
ing proteins, which include Ras, Rho, EGF Epidermal growth
and Rac, and GTP exchange mediators poietin, interferons, and a variety of
factor
such as Sos.6 Sos facilitates the conver- other hematopoietins signal via recep-
EGFR Epidermal growth
sion of GDP-bound Ras, the inactive factor receptor
tors belonging to the cytokine receptor
form of Ras, to GTP-bound Ras, the ER Estrogen receptor (CR) superfamily. Unlike the growth
active form of Ras. Ras proteins are only ERE Estrogen response factor RTKs, cytokine receptors do not
active in cellular signal transduction element have intrinsic kinase activity. In the
when they are localized to the inner ERK Extracellular signal cytokine receptor signaling pathway
surface of the plasma membrane after regulated kinase (Fig. 1, white), CRs associate with and
attachment of fatty acids. Activated Ras GPCR G-protein–coupled activate a class of related cytoplasmic
recruits Raf-1, a ‘‘MAP kinase kinase,’’ to receptor tyrosine kinases, the Janus kinases
the plasma membrane, initiating a serine/ Grb2 Growth factor (JAKs 1, 2, and 3). Activated JAKs
receptor binding phosphorylate a family of latent tran-
threonine kinase cascade that ultimately
protein 2 scription factors known as signal trans-
leads to phosphorylation of members of
IGF Insulin-like growth ducers and activators of transcription
the MAP kinase family.7 factor
How the MAP kinase signal crosses (STATs). STAT proteins form ho-
JAK Janus kinase
the nuclear envelope and affects gene modimers and heterodimers upon ty-
JNK c-jun N-terminal
expression is not fully understood. rosine phosphorylation. They then
kinase
However, MAP kinase can phosphory- MAP kinase Mitogen-activated
translocate to the nucleus, where they
late proteins, such as p90rsk, that can protein kinase directly activate the transcription of
translocate into the nucleus upon mi- MAPKK MAP kinase kinase responsive target genes.9
togen stimulation. In fact, several MAP (Raf ) Most cytokine receptors signal
kinase substrates are nuclear transcrip- MAPKKK MAP kinase kinase through the activation of the JAK/
tion factors, including p60TCF, c-jun, kinase (MEK) STAT pathway, with the most notable
c-myc, and the estrogen receptor (ER). MKP-1 MAP kinase phospha- exception being tumor necrosis fac-
tase tor-a (TNF-a). Activation of the JAK
Therefore, the nuclear translocation
PKC Protein kinase C kinases leads to cytokine receptor
of MAP kinase is consistent with a role
Ras Raous avian sarcoma phosphorylation, which serves as a
in signal transduction from the cyto-
proto-oncogene docking site for the SH2 domain con-
plasm to the nucleus. (GTPase protein)
tained in STAT proteins. The receptor-
Raf MAP kinase kinase
bound STATs can then be phosphory-
RTK Receptor tyrosine
Stress-Activated Protein Kinases kinase
lated and activated by JAKs. STATs
(SAPKs) SAPK Stress-activated pro- dissociate from the receptor, form
tein kinase head-to-tail dimers that translocate to
As with the MAP kinase cascades de- the nucleus, bind to specific DNA se-
SH2 Src-homology
monstrable in both mammalian and quences, and induce gene expression.9
domain 2
yeast cells, the stress-activated protein SH3 Src-homology
kinase (SAPK) pathways (Fig. 1, yel- domain 3
low) align themselves into a well- Sos Adaptor protein with NUCLEAR STEROID HORMONE
conserved, sequential cascade consist- SH2 domains that
ing of MAP kinase homologs (or SAPK/ links RTK to Ras
RECEPTORS
JNK/p38 MAP kinase) and MAP kinase c-Src Proto-oncogene that The actions of steroid hormones are
kinase homologs (or MKK/MEK/SEK/ encodes a mem- mediated through specific members of
JNKK).8 The activation of SAPKs re- brane-associated a large superfamily of nuclear recep-
lies on their phosphorylation at spe- tyrosine specific tors that function as ligand-activated
cific dual phosphorylation motifs, protein kinase transcription factors. These receptor
STAT Signal transducers
namely the sequences Thr-Pro-Tyr proteins share a common structural
and activators of
(TYP) of JNK and Thr-Glu-Tyr (T-Y-G) and functional organization, with dis-
transcription
of p38 MAP kinase. As predicted from tinct domains that are responsible for
TUTORIAL
ligand binding, DNA binding, and
transcriptional activation. Steroid re-
ceptors are composed of three key
functional domains: a hormone-inde-
pendent transcriptional activation
function domain (AF-1) located at the
N terminus, an internal DNA binding
domain (DBD) with its DNA-binding
zinc fingers, and a C-terminal hor-
mone binding domain (HBD). There
are various subdomains located within
the HBD and DBD that are critical to
the function of the receptor. For ex-
ample, located within the DBD is a
basic region containing a constitu-
tively active, hormone-independent,
nuclear localization signal, while a
hormone-dependent nuclear localiza-
tion signal is located in the HBD. Also
located in the HBD are a dimerization
domain, a heat shock protein-binding
region, and a hormone-dependent
transcriptional activation function
termed AF-2. Upon binding of ligand,
the conformation of the receptor
changes such that specific sites be-
come available for phosphorylation,
leading to activation of the AF-2 and
dimerization domains. Activated re-
ceptors then then form homo- or het-
erodimers that recognize and bind cis
elements in the upstream region of
responsive genes.10
Members of the steroid receptor
family are known to be phosphopro-
teins, and hormone-dependent phos-
phorylation has been observed for all
members of this family examined to
date. In the case of the estrogen recep-
tor (ER) (Fig. 1, red), there is a low
level of phosphorylation in the ab-
sence ligand, and the addition of li-
gand (17 b-estradiol) induces phos-
phorylation to about three- to fivefold.
This is important for activation of
transcription by the AF-1 domain. De-
letion and point mutation studies have
demonstrated that ligand binding in-
duces phosphorylation at multiple ser-
ine residues in the AF-1 region of the
ER, the most important being serine-
118, the phosphorylation of which is
essential for receptor transactivation.
Serine-167 is also phosphorylated in a
hormone-dependent manner and is im-
portant for DNA binding by the recep-
tor. In addition, certain tyrosine resi-
dues in the HBD of the ER have been
implicated in modulating hormone
binding.11
THE ANATOMICAL RECORD (NEW ANAT.) 45
CROSSTALK BETWEEN RECEPTOR
PATHWAYS
The presence of crosstalk between
GPCR, RTK, CR, and steroid receptors
suggests that interactions between
growth factors, polypeptide hormones,
cytokines, and steroid receptors work
together in a complex but coordinated
fashion to regulate cell physiology.
Regulation of SAPK Cascades
by Low-Molecular-Weight
G-Proteins and Ras-Activated
Kinases
Because of the role that Ras plays in
the upstream events initiating activa-
tion of the Raf/MEK/MAP kinase path-
way by growth factors, much interest
has centered on the involvement of
low-molecular-weight GTP-binding
proteins in the stress-related path-
ways, including not only Ras itself but
also the members of the Rho subfam-
ily. Data indicate that Rac and Cdc42
lie upstream in the JNK and p38 MAP
kinase cascades, and there is evidence
that Ras plays a role in the regulation
of SAPK activation. It has also been
found that expression of constitutively
active Ras, H-Ras, or D12 Ras results
in increased activity of JNK, while
JNK activation, in response to EGF, is
abrogated under conditions of expres-
sion of dominant-negative Ras.8
Various intracellular proteins have
been identified as potential targets for
the SAPKs, including a number of
transcription factors, such as c-jun,
activated transcription factor 2 (ATF2),
and CREB, which forms homo/het-
erodimer complexes that interact with
the activator protein-1 (AP-1) complex
to regulate gene expression. Early stud-
ies identified c-jun as a substrate for
JNK. Like JNK, p38 MAP kinase may
also modulate transcriptional events
by direct phosphorylation and activa-
tion of transcription factors.12,13
Cytokine Receptor Crosstalk
With Growth Factor Receptor
and Nuclear Receptor Pathways
There are several reports of JAKs phos-
phorylating and activating signaling
proteins normally associated with
growth factor signaling pathways. The
adapter molecule, Shc, is one putative
JAK target. Phosphorylated Shc forms
a complex with the adapter protein,
Grb2, which then binds and activates
the guanonucleotide-exchange factor,
Sos. This, in turn, leads to Ras activa-
tion followed by Raf-1 translocation to
the plasma membrane and activation.
Raf-1 initiates a signal to MEK1, thus
activating the MAP kinase cascade.
The MAP kinases in turn translocate
to the nucleus, phosphorylate and acti-
vate transcription factors, and regu-
late gene expression. There is also
evidence that JAK1 or JAK2 can di-
rectly phosphorylate Raf-1.14
Some of the cytokine receptors,
namely the interleukin-4 receptor
(IL4R), utilize the docking proteins,
IRS-1 and IRS-2, to mediate their sig-
naling. It is thought that JAKs are
capable of phosphorylating tyrosine
residues on IRS-1 and IRS-2. Tyrosine
phosphorylation forms docking sites
for proteins containing SH2 domains.
Some of these molecules are adapter
proteins, such as Grb2; other adapter
proteins indirectly couple to kinases,
such as the p85 regulatory subunit of
phosphotidyl-inositol-3 (PI-3) kinase;
others are protein-tyrosine phospha-
tases that remove phosphate modifica-
tions from tyrosines. The IRS family
of proteins was initially discovered as
intermediaries of insulin and IGF-1
signaling. Thus, if JAKs can phos-
phorylate IRS proteins, they may be
able to activate signaling of these
growth factor receptors in the absence
of ligand. Growth factor receptors
themselves are another potential tar-
get of JAKs. Stimulation of the growth
hormone (GH) receptor, a member of
the class I CR family, has been shown
to lead to JAK phosphorylation and
activation of the epidermal growth
factor receptor (EGFR). The unli-
ganded EGFR can then signal through
the Grb2/Sos = Ras = Raf-1 = MEK1
= MAP kinase pathway. This may
provide a mechanism for the direct
proliferative action of GH.9
GPCR Interactions With MAP
Kinase Cascade, cAMP
Pathways, and Nuclear
Transcription Factors
G-protein–coupled receptor–mediated
events may be regulated by two classi-
cal Ga effectors, adenylate cyclase (AC)
and phospholipase Cg (PLCg). Activa-
tion of AC by Ga causes an increase in
46 THE ANATOMICAL RECORD (NEW ANAT.)
cytosolic cAMP that can then bind to
the regulatory subunits of protein ki-
nase A (PKA), leading to release and
activation of the catalytic subunits.
PKA can then modulate GPCR activity
by phosphorylating the receptor and
causing desensitization or switching
of the activated abg isotype. However,
PKA has several other substrates, in-
cluding Raf-1, which is prototypically
activated by growth factor receptor
tyrosine kinases, and is an intermedi-
ary in MAP kinase activation. Raf-1
phosphorylation by PKA has been re-
ported to lead to a decrease in Raf-1
activity and attenuation of growth fac-
tor–induced MAP kinase activity. An-
other target of PKA is the transcrip-
tion factor, CREB. Phosphorylation of
CREB by PKA causes activation
whereby CREB can bind and induce
the expression of genes containing the
CRE upstream enhancer sequence. The
dual-specificity phosphatase, MAP ki-
nase phosphatase (MKP-1), which is
specific for MAP kinase family mem-
bers, contains a putative CRE site.
Thus, PKA activity may induce MKP-1
expression, which then dephosphory-
lates and inactivates/activates MAP ki-
nases.4
The Gbg subunit of GPCRs is also
capable of signal transduction; how-
ever, much less is known about the
mechanism or effector molecules. In
cardiac myocytes, the bg subunit can
bind to and activate L-type calcium
channels, causing an influx of calcium
into cells. Downstream effects might
then be similar to those described
above for the IP3-mediated increase in
intracellular calcium. Recently, it was
shown that the bg subunit is able to
bind and activate growth factor recep-
tors, specifically EGFR. Activation of
EGFR by the bg subunit is believed to
be mediated through an activation of
MAP kinase via Grb2/Sos = Ras = Raf-1
= MEK1.15
Crosstalk From the Membrane
to the Nucleus
It has become apparent that many
transcription factors contain activa-
tion domains whose interaction with
transcriptional complex proteins or
coactivators is facilitated by phosphor-
ylation. For example, peptide growth
factors are able to activate nuclear
transcription factors through phos-
phorylation via specific kinases. Ste-
roid-induced or extracellular ligand-
induced phosphorylation of nuclear/
steroid hormone receptors may in part
regulate transcriptional activity. For
example, phosphorylation of the ER
on both serine and tyrosine residues
has been observed by several laborato-
ries. Phosphorylation of N-terminal
serine residues on the ER influences
the receptor’s transcriptional activity.
These N-terminal serine residues may
be candidate phosphorylation sites for
peptide growth factor–mediated regu-
lation of ER transcriptional (AF-1) ac-
tivity.11 For example, serine-118 can
be phosphorylated by EGF or IGF-1
and lies within a consensus MAP ki-
nase recognition motif.16 Both in vitro
and in vivo activation of MAP kinase
has been shown to phosphorylate the
ER and induce ligand-independent
transcriptional activity. Similarly, ser-
ine-236 has been shown to be phos-
phorylated by protein kinase A (PKA),
resulting in enhanced gene expres-
sion. Thus, stimuli that increase cAMP
and subsequently PKA (such as
GPCRs) can modulate the transcrip-
tional activity of the ER.17 In addition
to the ER, the progesterone receptor
has been shown to be activated inde-
pendent of ligand by cAMP and IGF-1.
The precise signal transduction path-
ways that mediate peptide growth fac-
tor–induced transcription through the
ER and other steroid hormone recep-
tors have yet to be completely eluci-
dated; however, recent reports suggest
that the particular signaling pathways
involved may very well be cell-type-
specific.
In addition to ligand-independent
activation of steroid hormone recep-
tors, activation of other signaling cas-
cades can affect ligand-dependent
function. Coactivators and corepres-
sors are essential for steroid receptor
modulation of gene expression. These
large proteins form complexes with
the activated steroid receptor family.
Some of these proteins possess intrin-
sic enzyme activity or associate with
proteins that have histone acetylase/
deacetylase activity. Histone deacetyla-
tion is thought to be important in
chromatin remodeling and access to
DNA by transcription factors. One of
the proteins that coactivates this com-
plex is CBP/p300, which contains his-
tone deacetylase activity and is re-
TUTORIAL
quired for gene expression by certain
steroid receptors. CBP/p300 is also
used by other transcription factors,
namely AP-1 and STATs, in regulating
gene expression.18 Since the quantity
of CBP/p300 in the cell is limited, its
avalibility is one of the rate-limiting
steps in gene expression. Thus, depend-
ing on the hormonal, growth factor,
and cytokine mileu and the order and
magnitude of the pathways stimu-
lated, a ligand may or may not regu-
late gene expression or may demon-
strate different degrees of gene
regulatory activity at different points
in time. For example, estradiol treat-
ment of MCF-7 breast cancer cells
stimulates estrogen response element
(ERE)–mediated gene transcription;
however, pretreatment of these cells
with IGF-1 followed by estradiol causes
a decrease in ERE-mediated gene tran-
scription. This may occur because
stimulation by IGF-1 results in the
sequestration of the available CBP/
p300 to induce AP-1–mediated gene
expression, and CBP/p300 is thus un-
available for ER-mediated gene expres-
sion.19
It has been observed that protein
kinase activators enhance the tran-
scriptional activity of the ER and that
changes in the cellular phosphoryla-
tion state are important in determin-
ing the biological effectiveness of the
estrogen-occupied receptor. Activa-
tion of PKC-mediated pathways can
lead to the enhanced expression and
phosphorylation of the proto-onco-
genes, c-fos and c-jun. Fos/jun het-
erodimers or jun/jun homodimers rec-
ognize and bind AP-1 sites in DNA to
modulate transcription of steroid re-
ceptor–mediated genes. In the ovalbu-
min promoter, a half-palindromic ERE
is coactivated by ER and fos/jun onco-
proteins. In addition, it has been
shown that activators of PKA and PKC
markedly synergize with estradiol in
ER-mediated transcriptional activa-
tion and that this transcriptional syn-
ergism shows cell-type and promoter
specificity.20
In addition to the two mechanisms
mentioned above, nuclear receptor ac-
tivity can be mediated by membrane
receptor activity at the DNA level. For
example, stimulation of PKC/PKA and
associated stimulation of cAMP leads
to the activation and binding of CREB
to its DNA binding site, CRE. This
TUTORIAL
enhancer element is then able to modu-
late the expression of given genes in
concert with the activation of other
enhancer and/or suppressor elements.
Thus, signaling pathways such as the
JNKs, SAPKs, or GPCRs which modu-
late cAMP levels could secondarily
modulate the transcription of certain
sets of genes. Another example of this
type of pathway crosstalk would be
between the AP-1 (fos/jun) pathway
and the ER pathway. Estrogen is a
potent mitogen for breast epithelial
cells, and, as part of this mitogenic
process, estrogen stimulation results
in a rapid induction of c-fos expres-
sion. Enhanced c-fos expression will
alter the ratio of jun/jun homodimers
to fos/jun heterodimers, resulting in
differential modulation of the AP-1
enhancer element located in the up-
stream regulatory region of many
genes. Recently, an AP-1 site that func-
tions as a strong enhancer element has
been described in the upstream regula-
tory region of the ER gene. Thus,
modulation of c-fos expression by es-
trogen can differentially modulate
AP-1 activity that can in turn modu-
late ER gene expression. Given that
upstream regulatory regions of many
genes may contain ERE, CRE, and
AP-1 sites, the sequence of receptor
activation may play an important role
in the fine modulation of gene expres-
sion.
Feedback Mechanisms From
the Nucleus to Upstream
Signaling Pathways
The exact feedback pathways from
nuclear transcription factors/steroid
receptors to membrane receptors re-
main unclear. However, since almost
all actions of steroid receptors are due
to their function as transcription fac-
tors, it is likely that feedback occurs
through modulation of gene expres-
sion. Steroid receptors can repress the
expression of either signaling mol-
ecules or growth factors. Glucocorti-
coids suppress interleukin-2 expres-
sion in T cells, which is necessary for
T-cell proliferation, activation, and
chemotaxis. Additionally, steroid re-
ceptors could induce the expression of
genes that actively attenuate signal-
ing, either as phosphatases or nonen-
zymatic proteins that sterically block
THE ANATOMICAL RECORD (NEW ANAT.) 47
activation sites or compete with
adapter molecules to disrupt signaling
complexes or pathways. In addition,
two steroid hormones, estradiol and
retinoic acid, have been reported to
modulate intracellular cAMP levels.
For example, retinoic acid has been
found to significantly inhibit prosta-
glandin E2-induced cAMP accumula-
tion in a dose-dependent manner in
osteoblast-like cells. A final mecha-
nism of feedback regulation is direct
modulation of growth factor (i.e.,
EGFR), GPCR, or CR gene expression,
thereby reducing or increasing the
available receptors on the cell sur-
face.21
Although the interactions of cell sig-
naling pathways are complex and to
some extent confusing, and even
though new pathways of integration
are still being identifed, we must begin
to try to piece together some general
scheme of the pathways by which dif-
ferent receptor or cell signaling path-
ways crosstalk. To meet this challange,
several biological tenets must be inte-
grated into any formal model for sig-
nal transduction crosstalk mecha-
nisms. First, specificity must apply, as
in the case of high-affinity ligand-
receptor interactions. However, speci-
ficity cannot be directly applied to
whole ensembles of molecules such as
those that transduce signals from the
membrane to the nucleus or from the
nucleus to the membrane or cyto-
plasm. In addition, signaling path-
ways are vested with multiple func-
tions, including signal amplification
and signal damping, and thereby coor-
dinate input through crosstalk. Sec-
ond, through evolution, a patchwork
of restriction mechanisms has arisen
from coupling together available com-
ponents by natural selection. Further-
more, it must be remembered that
although the interactions between
components that signal from and to
the nucleus are quantitative in nature,
most of our experimental approaches
are semiquantitative or merely qualita-
tive. It is clear from the literature that
various signaling pathways do
crosstalk with each other to both am-
plify and dampen the activity of extra-
cellular and intracellular signals. Al-
though it is easy to become mired in
the minutia of these signaling path-
ways, it is essential that, from time to
time, we take inventory of our knowl-
edge and that we look at the big pic-
ture of how crosstalk between the
various pathways leads to an inte-
grated control of both cell and organ
physiology.
ACKNOWLEDGMENTS
I thank the past and current members
of this laboratory for their excellent
contributions, which have formed the
basis for part of this review.
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