Tài liệu Báo cáo khoa học: Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells pptx

Endogenous expression and protein kinase A-dependent
phosphorylation of the guanine nucleotide exchange
factor Ras-GRF1 in human embryonic kidney 293 cells
Jens Henrik Norum
1
, Trond Me
´
thi
1
, Raymond R. Mattingly
2
and Finn Olav Levy
1
1 Department of Pharmacology, University of Oslo, Norway
2 Department of Pharmacology, Wayne State University, Detroit, MI, USA
Introduction
Signals mediated through receptor tyrosine kinases [1]
and G-protein-coupled receptors (GPCRs) can induce
the activation of intracellular cascades such as the
mitogen-activated protein (MAP) kinase – also called
extracellular signal-regulated kinase (ERK) – cascade.
The serine ⁄ threonine kinases ERK1 and ERK2 are
activated by dual phosphorylation by the MAP kinase
kinase, MEK, which becomes phosphorylated and
activated by MEK kinases of the Raf family. All
three Raf isoforms [A-Raf, B-Raf and Raf-1 (C-Raf)]
Keywords
5-HT
7
, cAMP, ERK, GEF, serotonin
Correspondence

F. O. Levy, Department of Pharmacology,
University of Oslo, PO Box 1057 Blindern,
N-0316 Oslo, Norway
Fax: +47 22840202
Tel: +47 22840237 or +47 22840201
E-mail:
(Received 2 December 2004, revised 1
February 2005, accepted 10 March 2005)
doi:10.1111/j.1742-4658.2005.04658.x
We have previously reported the Ras-dependent activation of the mitogen-
activated protein kinases p44 and p42, also termed extracellular signal-
regulated kinases (ERK)1 and 2 (ERK1 ⁄ 2), mediated through G
s
-coupled
serotonin receptors transiently expressed in human embryonic kidney
(HEK) 293 cells. Whereas G
i
- and G
q
-coupled receptors have been shown
to activate Ras through the guanine nucleotide exchange factor (GEF)
called Ras-GRF1 (CDC25
Mm
) by binding of Ca
2+
⁄ calmodulin to its
N-terminal IQ domain, the mechanism of Ras activation through G
s
-cou-
pled receptors is not fully understood. We report the endogenous expres-

sion of Ras-GRF1 in HEK293 cells. Serotonin stimulation of HEK293
cells transiently expressing G
s
-coupled 5-HT
7
receptors induced protein
kinase A-dependent phosphorylation of the endogenous human Ras-GRF1
on Ser927 and of transfected mouse Ras-GRF1 on Ser916. Ras-GRF1
overexpression increased basal and serotonin-stimulated ERK1 ⁄ 2 phos-
phorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking
(Ser916Asp ⁄ Glu) phosphorylation did not alter these effects. However, the
deletion of amino acids 1–225, including the Ca
2+
⁄ calmodulin-binding IQ
domain, from Ras-GRF1 reduced both basal and serotonin-stimulated
ERK1 ⁄ 2 phosphorylation. Furthermore, serotonin treatment of HEK293
cells stably expressing 5-HT
7
receptors increased [Ca
2+
]
i
, and the sero-
tonin-induced ERK1 ⁄ 2 phosphorylation was Ca
2+
-dependent. Therefore,
both cAMP and Ca
2+
may contribute to the Ras-dependent ERK1 ⁄ 2 acti-
vation after 5-HT

7
receptor stimulation, through activation of a guanine
nucleotide exchange factor with activity towards Ras.
Abbreviations
5-HT, 5-hydroxytryptamine (serotonin); CaM, calmodulin; EGF, epidermal growth factor; Epac, exchange protein directly activated by cAMP;
ERK, extracellular signal-regulated kinase; GEF, guanine nucleotide exchange factor; GPCR, G protein-coupled receptor; H89,
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; HEK, human embryonic kidney; HRP, horseradish peroxidase;
MAP, mitogen-activated protein; MEK, mitogen-activated protein ⁄ extracellular signal-regulated kinase kinase; PKA, protein kinase A; Sos1,
son of sevenless 1.
2304 FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS
expressed in mammalian cells may become activated
by members of the Ras family of small G proteins.
The activity of Ras proteins is under tight control of
several classes of guanine nucleotide exchange factors
(GEFs) and GTPase-activating proteins. Mammalian
Son of sevenless 1 (Sos1) is a ubiquitous Ras GEF and
activates Ras following the stimulation of receptor
tyrosine kinases, e.g. the epidermal growth factor
(EGF) receptor [1]. Various GPCRs can also induce
Ras activation via several classes of GEFs [2,3].
Activation of phospholipase C through G
q
-coupled
receptors, with subsequent increased levels of inositol-
1,4,5-trisphosphate, diacylglycerol and free intracellular
Ca
2+
, can activate Sos1 through a cascade that
includes the proline-rich tyrosine kinase, Pyk2, Src and
Grb2 [4,5], as well as Ras GEFs of the Ras-GRP (cal-

DAG-GEF) family, through binding of Ca
2+
⁄ calmo-
dulin (CaM) and diacylglycerol [6]. Ras-GRF1, also
called CDC25
Mm
[7,8], is another major GEF with
activity towards Ras. Ras-GRF1 mediates activation
of Ras subsequent to the stimulation of G
i
- and G
q
-
coupled receptors [8,9].
The main mechanism for the activation of Ras-
GRF1 is the binding of Ca
2+
⁄ CaM to the N-terminal
IQ motif [10]. We have previously shown that the
treatment of NIH3T3 and COS-7 cells with carbachol
[9] and lysophosphatidic acid [11], activating both
G
q
- and G
i
-coupled receptors, induces the activation
and phosphorylation of Ras-GRF1. Furthermore,
Ras-GRF1 is also heavily phosphorylated upon agon-
ist activation of GPCRs, but the exact role of these
phosphorylations is not fully understood. Protein kin-

ase A (PKA) is one of probably several kinases that
can induce the phosphorylation of Ras-GRF1 [12,13].
The residues Ser916 and Ser898 in the mouse and rat
sequences, respectively, are homologous PKA phos-
phorylation sites [14]. Although forskolin-induced
phosphorylation of Ser916 is not sufficient to activate
wild-type Ras-GRF1, a recombinant version of Ras-
GRF1, with a mutated phosphorylation site (Ser916-
Ala), has been shown to have reduced activity
towards Ras both in vitro [12] and in an assay of
Ras-dependent outgrowth of neurites from PC12 cells
[14]. These results indicate that even though phos-
phorylation of Ser916 may contribute to stimulation
of the Ras-GEF activity of Ras-GRF1, cAMP-
dependent phosphorylation alone is not sufficient to
activate Ras-GRF1.
Ras-GRF1 is mainly expressed in brain tissue [15–
17], but expression of Ras-GRF1 mRNA has also been
reported in some other tissues and non-neural cell lines
[18]. Several murine Ras-GRF1 cDNAs, encoding
proteins of different molecular mass (from 54 to
140 kDa), have been identified [17,19]. The smaller iso-
forms correspond to N-terminal deletions of the full-
length 140 kDa protein. The physiological role of the
guanine nucleotide exchange activity of the truncated
forms is not known as they are missing the Ca
2+
⁄
CaM-binding IQ domain that is involved in the activa-
tion of Ras-GRF1.

Stimulation of all the splice variants of the G
s
-coupled
serotonin receptor 5-hydroxytryptamine
7
(5-HT
7
)
increases intracellular levels of the second messenger
cAMP [20], resulting in several intracellular effects, e.g.
activation of cAMP-dependent protein kinase (PKA)
and exchange proteins directly activated by cAMP
(Epacs), GEFs specific for Rap [21]. In rat adrenal glo-
merulosa cells, stimulation of the 5-HT
7
receptor also
induces the increased free intracellular Ca
2+
concentra-
tion ([Ca
2+
]
i
) through the low-voltage-activated T-type
Ca
2+
channels [22,23]. We have recently shown that
serotonin treatment of human embryonic kidney
(HEK)293 cells transiently expressing either one of the
G

s
-coupled serotonin receptors 5-HT
4(b)
or 5-HT
7(a)
induces ERK1 ⁄ 2 phosphorylation [24]. Although both
Ras and Rap1 were activated, only Ras was involved in
the pathway inducing ERK1 ⁄ 2 phosphorylation, which
also involved Raf1 and MEK downstream of Ras. How-
ever, in PC12 cells, 5-HT
7
-mediated Ca
2+
-independent
and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-
sulfonamide dihydrochloride (H89)-insensitive ERK1 ⁄ 2
phosphorylation has been reported to be enhanced by
the overexpression of Epac and mimicked by a cAMP
analogue stimulating both PKA and Epac, but not by
an Epac-specific cAMP analogue [25]. The differences in
H89 sensitivity and possible signalling pathways
involved may reflect cell-type variations in the ERK1 ⁄ 2
phosphorylation mediated through G
s
-coupled sero-
tonin receptors.
The mechanism of Ras activation through G
s
-cou-
pled receptors is not fully understood. In the present

study, we show endogenous expression of the
Ca
2+
-dependent 140 kDa and shorter isoforms of Ras-
GRF1 in HEK293 cells, as well as cAMP ⁄ PKA-
dependent phosphorylation of Ras-GRF1 associated
with ERK1 ⁄ 2 phosphorylation following stimulation
of transfected 5-HT
7
receptors. However, mutating
Ser916 of Ras-GRF1 to alanine, aspartic acid or gluta-
mic acid did not alter the Ras-GRF1-induced ERK1 ⁄ 2
phosphorylation. We confirm 5-HT
7
-mediated [Ca
2+
]
i
increase and show Ca
2+
dependence of serotonin-
induced ERK1 ⁄ 2 phosphorylation and a mandatory
role of the Ca
2+
⁄ CaM-binding IQ domain in Ras-
GRF1-stimulated ERK1 ⁄ 2 phosphorylation. Thus,
both cAMP and Ca
2+
may contribute to Ras-depend-
ent ERK1 ⁄ 2 activation following stimulation of the

J. H. Norum et al. Ras-GRF1 and Ras-dependent ERK activation in HEK293
FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS 2305
5-HT
7
receptor, by activating a guanine nucleotide
exchange factor with activity towards Ras.
Results
HEK293 cells express the guanine nucleotide
exchange factor Ras-GRF1
The guanine nucleotide exchange factor Ras-GRF1 is
mainly expressed in neurones of the central nervous
system, although it has also been reported to be
expressed in some other tissues [18,26]. To investigate
whether Ras-GRF1 plays a role in the activation of
Ras ⁄ ERK signalling in HEK293 cells, we first used
immunoprecipitation analysis and RT-PCR to detect
whether Ras-GRF1 protein and mRNA, respectively,
were expressed in our HEK293 cells. The proteins im-
munoprecipitated from HEK293 cell lysates, by using
a polyclonal antibody raised against a peptide mapping
to the C terminus of the rat Ras-GRF1 sequence, were
separated on SDS⁄ PAGE (6% gel) and visualized on
western blots probed with another polyclonal antibody
raised against the C terminus of the human Ras-GRF1
sequence. A protein of  140 kDa was detected in
immunoprecipitates from HEK293 cells (Fig. 1A), but
was not present in control immunoprecipitations. In
whole-cell lysates from HEK293 cells, both full-length
140 kDa Ras-GRF1 and shorter isoforms, of  110, 95
and 60 kDa, were detected on western blots with

anti-(Ras-GRF1) Ig (Fig. 2A, right panel). Preabsorb-
ing the Ras-GRF1 antibody with a blocking peptide
prevented the antibody from recognizing any of the
Ras-GRF1 isoforms (data not shown).
cDNA to HEK293 cell mRNA was used as the sub-
strate in PCR reactions, as described in the Experi-
mental procedures. The primer pairs specific for the
A
B
Fig. 1. Human embryonic kidney (HEK)293 cells express Ras-GRF1.
(A) Paramagnetic beads coated with anti-(Ras-GRF1) Ig (# sc-224)
were used to immunoprecipitate Ras-GRF1 from the HEK293 cell
lysate. The precipitated proteins were separated on 6% SDS ⁄ PAGE
and electroblotted over to poly(vinylidene difluoride) membranes
before probing with polyclonal Ras-GRF1 antibodies (# sc-863).
(B) cDNA produced from mRNA isolated from HEK293 cells was
used as the substrate in RT-PCR with primer pairs specific for
human Ras-GRF1. Primer pairs: lane 2, ON359 and ON360; lane 3,
ON357 and ON358; and lane 4, ON361 and ON360. The PCR prod-
ucts and a DNA size marker, lane 1, were separated on agarose
gels. The expected sizes of the PCR products, in bp, are indicated
to the right.
A
B
C
D
Fig. 2. Serotonin induces phosphorylation of Ras-GRF1 through the
5-hydroxytryptamine
7(a)
(5-HT

7(a)
) receptor, and HA-Ras-GRF1 indu-
ces extracellular signal-regulated kinase (ERK)1 ⁄ 2 activation.
Human embryonic kidney (HEK)293 cells cotransfected with
5-HT
7(a)
receptor and empty or HA-Ras-GRF1 vector, as indicated,
were treated with vehicle or 10 l
M serotonin for the indicated peri-
ods of time. The control, C, was treated with vehicle (10 l
M HCl)
for 5 min. Proteins were separated on 6% (A, B and D) or 10%
(C) SDS ⁄ PAGE and electroblotted over to poly(vinylidene difluoride)
membranes before probing with antibodies. (A) Western blots were
probed with phosphospecific Ras-GRF1 (pRas-GRF1; left panel) or
anti-(Ras-GRF1) Igs (Ras-GRF1; right panel) to confirm equal load-
ing. (B) Western blot of cell lysates of HEK293 cells cotransfected
with the 5-HT
7(a)
receptor and HA-Ras-GRF1 were incubated with
anti-(pRas-GRF1) Ig (upper panel) or anti-(Ras-GRF1) Ig (lower panel)
to confirm equal loading. (C) The same samples as in (A) and (B),
but separated on 10% SDS ⁄ PAGE, were probed with phosphospe-
cific ERK1 ⁄ 2 antibodies (pERK1 ⁄ 2; upper panel) and subsequently
with ERK1 ⁄ 2 antibodies (ERK1 ⁄ 2; lower panel), to confirm equal
loading. (D) Non-transfected HEK293 cells were treated with or
without 10 n
M epidermal growth factor (EGF) for 5 min. The pro-
teins were separated, blotted and probed with antibodies as in (A).
Ras-GRF1 and Ras-dependent ERK activation in HEK293 J. H. Norum et al.

2306 FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS
human Ras-GRF1 nucleotide sequence (NM_002891,
GI:24797098) gave PCR products of expected size
(Fig. 1B). The primer sequences are located at the
5¢ end (ON361, ON360 and ON359) and in the middle
(ON357 and ON358) of the human Ras-GRF1 nucleo-
tide sequence. Sequencing of the purified PCR prod-
ucts confirmed sequence identity with cDNA encoding
the human 140 kDa Ras-GRF1 (data not shown).
Taken together, these mRNA and protein data demon-
strate that the full-length 140 kDa Ras-GRF1 protein
is endogenously expressed in the HEK293 cells used
for this study, and that truncated forms of Ras-GRF1
may also be present.
Serotonin induces phosphorylation of Ras-GRF1
through 5-HT
7
receptors
The serine residue at position 916 in mouse Ras-GRF1
is a PKA phosphorylation site both in vitro [12] and
in vivo [14], and the corresponding human residue is
serine 927. We therefore used a polyclonal antibody
that was generated against a synthetic phosphopeptide
analogous to the Ser916 phosphorylation site, and
which has previously been shown to recognize mouse
and rat Ras-GRF1 when they are phosphorylated at
this residue [14], to test whether serotonin may stimu-
late phosphorylation of Ras-GRF1 in HEK293 cells
that express 5-HT
7

receptors. HEK293 cells transfected
with 5-HT
7(a)
receptors alone, or cotransfected with
the HA-tagged mouse Ras-GRF1 (HA-Ras-GRF1),
were treated with 10 lm serotonin for the indicated
periods of time (Fig. 2). Serotonin treatment increased
the phosphorylation of the endogenous 140 kDa and
 60 kDa isoforms of Ras-GRF1 (Fig. 2A, left panel)
and of recombinant HA-Ras-GRF1 (Fig. 2B, upper
panel). Phosphorylation of ERK1 ⁄ 2 in the same sam-
ples was fully induced after 3 min of treatment with
10 lm serotonin (Fig. 2C). Furthermore, both basal
and serotonin-induced phosphorylation of ERK1 ⁄ 2
was increased in cells cotransfected with HA-Ras-
GRF1 and 5-HT
7(a)
receptor, compared to cells trans-
fected with receptor only (Fig. 2C).
The EGF receptor induces activation of Ras
through a GEF, called Sos1, in a Ca
2+
-independent
manner. Treatment of HEK293 cells with 10 nm EGF
for 5 min resulted in ERK1 ⁄ 2 phosphorylation
(Fig. 6D) but not in phosphorylation of endogenous
Ras-GRF1 at the site recognized by the antibody
directed against Ras-GRF1 phosphorylated at Ser916 ⁄
927 (Fig. 2D). This indicates that ERK1 ⁄ 2 activation
induced by EGF does not increase the phosphoryla-

tion of endogenously expressed Ras-GRF1 on Ser927.
EGF has similarly been reported not to increase
phosphorylation of endogenous Ras-GRF1 in rat
brain [9].
Serotonin-induced phosphorylation of Ras-GRF1
is dependent on cAMP and PKA
Serotonin increases adenylyl cyclase activity in
HEK293 cells expressing the human G
s
-coupled sero-
tonin receptor 5-HT
7
[27]. Forskolin increases aden-
ylyl cyclase activity and induces the phosphorylation
of Ser916 in the mouse Ras-GRF1 sequence [12] and
of Ser898 in the rat sequence [14]. To test whether
serotonin stimulated Ras-GRF1 phosphorylation
through PKA, HEK293 cells were cotransfected with
5-HT
7(a)
receptors, HA-Ras-GRF1 and either empty
vector or the human phosphodiesterase hPDE4D2,
which indirectly reduces PKA activity by reducing
cAMP levels. The serotonin-induced phosphorylation
of HA-Ras-GRF1 was essentially abolished and
ERK1 ⁄ 2 phosphorylation was reduced in cells
cotransfected with hPDE4D2 (Fig. 3A). The phos-
phorylation of overexpressed HA-Ras-GRF1 was also
eliminated in cells incubated with 20 lm H89, an
inhibitor of PKA but also of other kinases [28], for

25 min prior to treatment with 10 lm serotonin
(Fig. 3B). The serotonin-induced ERK1 ⁄ 2 phosphory-
lation was concomitantly reduced, as expected based on
the results of our previous publication [24]. Cotrans-
fection of HEK293 cells with the PKA inhibitor protein
kinase inhibitor, in addition to 5-HT
7(a)
receptors and
HA-RasGRF1, also reduced the serotonin-induced
phosphorylation of recombinant HA-Ras-GRF1, as
well as ERK1⁄ 2 phosphorylation (not shown). Phos-
phorylation of the endogenously expressed 140 kDa
and  60 kDa isoforms of Ras-GRF1 was increased
following stimulation with serotonin. The  60 kDa iso-
form of Ras-GRF1 seems to be expressed at a higher
level than the 140 kDa isoform. The serotonin-induced
increase in phosphorylation of both isoforms was
reduced by the coexpression of hPDE4D2 with 5-HT
7
receptors (Fig. 3C). This was also the case for ERK1 ⁄ 2
phosphorylation (Fig. 3D).
Phosphorylation of Ser916 is neither necessary
nor sufficient for Ras-GRF1-mediated
phosphorylation of ERK1 ⁄ 2
To investigate the potential role of phosphorylation
at Ser916 ⁄ Ser927 of Ras-GRF1 in 5-HT
7(a)
receptor-
dependent ERK1 ⁄ 2 activation, we compared the
activities of wild-type Ras-GRF1 to proteins that

had single amino acid substitutions at Ser916. We
also used the mutants to verify the specificity of the
J. H. Norum et al. Ras-GRF1 and Ras-dependent ERK activation in HEK293
FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS 2307
phosphoRas-GRF1 antibody. The antibody to phos-
phoSer916-Ras-GRF1 was developed against a syn-
thetic phosphopeptide corresponding to the residues
surrounding Ser916 of mouse Ras-GRF1 and had
previously been shown to be unreactive with a Ras-
GRF1 Ser916Ala mutant protein that was expressed
in COS-7 or PC12 cells [14]. The antibody did not
recognize HA-Ras-GRF1 proteins mutated at the
Ser916 residue to alanine, aspartic acid or glutamic
acid and expressed in HEK293 cells (Fig. 4A). Inter-
estingly, neither inhibiting phosphorylation of Ser916
by mutating the amino acid to alanine, nor poten-
tially mimicking it by mutation to aspartic acid or
glutamic acid, influenced the ability of recombinant
HA-Ras-GRF1 to induce phosphorylation of
ERK1 ⁄ 2 in HEK293 cells (Fig. 4B). These results
suggest that the phosphorylation of Ras-GRF1 at
this residue may be neither necessary nor sufficient
to mediate stimulation of ERK1 ⁄ 2 activation in
HEK293 cells.
An intact N-terminal region is required for
Ras-GRF1 to potentiate ERK1/2 activation
The role of calcium in the phosphorylation of ERK1 ⁄ 2
induced by Ras-GRF1 was addressed by cotransfecting
HEK293 cells with 5-HT
7(a)

receptors and Ras-GRF1-
D1-225 (i.e. lacking the PH1-, coiled-coil and IQ
domains). Cotransfection of HEK293 cells with this
truncated form of Ras-GRF1 did not increase the basal
or serotonin-induced phosphorylation of ERK1 ⁄ 2 com-
pared to cells transfected with the receptor only
(Fig. 4B). Serotonin treatment did, however, increase
the phosphorylation of Ras-GRF1-D1-225 on Ser916
(Fig. 4A).
Serotonin increases [Ca
2+
]
i
through 5-HT
7
receptors
We have previously shown that the G
s
-coupled sero-
tonin receptors 5-HT
4(b)
and 5-HT
7(a)
induce phos-
A
B
C
D
Fig. 3. Serotonin-induced Ras-GRF1 and ext-
racellular signal-regulated kinase (ERK)1 ⁄ 2

phosphorylation is dependent on protein kin-
ase A (PKA) ⁄ cAMP. (A) Human embryonic
kidney (HEK)293 cells cotransfected with
the 5-hydroxytryptamine
7(a)
(5-HT
7(a)
) recep-
tor, HA-Ras-GRF1, and either with or with-
out hPDE4D2, were treated with 10 l
M
5-HT for 5 min. (B) HEK293 cells cotrans-
fected with 5-HT
7(a)
receptor and HA-Ras-
GRF1 were treated with or without 20 l
M
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquin-
olinesulfonamide dihydrochloride (H89) for
25 min prior to treatment with or without
10 l
M serotonin for 5 min. (C) HEK293 cells
cotransfected with the 5-HT
7(a)
receptor and
empty vector or hPDE4D2, as indicated,
were treated with 10 l
M serotonin for
5 min. (D) The same samples as in (C) were
assayed for ERK1 ⁄ 2 phosphorylation by

SDS ⁄ PAGE (10% gel) and the western blot
was probed with phosphospecific ERK1 ⁄ 2
antibodies (pERK1 ⁄ 2; upper panel) and then
with ERK1 ⁄ 2 antibodies (ERK1 ⁄ 2; lower
panel), to confirm equal loading. The pro-
teins were separated by SDS ⁄ PAGE (6%
gel) for Ras-GRF1 and by SDS ⁄ PAGE (10%
gel) for ERK1 ⁄ 2 and electroblotted to
poly(vinylidene difluoride) membranes. The
membranes were probed with antibodies,
as indicated.
Ras-GRF1 and Ras-dependent ERK activation in HEK293 J. H. Norum et al.
2308 FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS
phorylation of ERK1 ⁄ 2 through a Ras-dependent
mechanism [24]. The two other known human 5-HT
7
receptor splice variants (5-HT
7(b)
and 5-HT
7(d)
) also
induce phosphorylation of ERK1 ⁄ 2 through a Ras-
dependent mechanism (data not shown). Therefore, in
this respect we consider the different 5-HT
7
splice vari-
ants to behave similarly when expressed in HEK293
cells. HEK293 cells stably expressing the 5-HT
7(b)
receptor (KB1 cells) were used to determine whether

serotonin can increase [Ca
2+
]
i
through human 5-HT
7
receptors. Treatment of the KB1 cells with 10 lm sero-
tonin resulted in a rapid, transient increase in [Ca
2+
]
i
,
with a maximum of 40–60% above the basal level,
whereas there was no effect of vehicle (10 lm HCl;
Fig. 5). To establish that the effect was mediated
through the 5-HT
7(b)
receptors, nontransfected
HEK293 cells were subjected to the same treatment;
no effect of serotonin on [Ca
2+
]
i
was detected. The
serotonin-induced increase in [Ca
2+
]
i
was abolished by
the calcium influx inhibitor, carboxyamido-triazole

(CAI) (20 lm), but not by vehicle control (dimethyl-
sulfoxide) (Fig. 5, inset). These results indicate that
serotonin (10 lm) can increase [Ca
2+
]
i
through the
human G
s
-coupled 5-HT
7
receptors in HEK293 cells.
The exact mechanism for the serotonin-mediated
increase in Ca
2+
levels is not known.
Phosphorylation of ERK1/2, mediated through
5-HT
7
receptors, is dependent on Ca
2+
Transiently transfected HEK293 cells were incubated
with CAI (20 lm) for 25 min prior to 5 min of treat-
A
B
Fig. 4. Mutation of Ser916 of mouse Ras-GRF1 does not alter the
activation of extracellular signal-regulated kinase (ERK)1 ⁄ 2 but dele-
tion of amino acids 1–225 blocks the stimulatory effect of Ras-
GRF1. Human embryonic kidney (HEK)293 cells transfected with
the 5-hydroxytryptamine

7(a)
(5-HT
7(a)
) receptor and empty vector or
with Ras-GRF1, Ras-GRF1Ser916Ala, Ras-GRF1Ser916Asp, Ras-
GRF1Ser916Glu or Ras-GRF1-D1-225, were treated with or without
10 l
M serotonin for 5 min. (A) Western blots of SDS ⁄ PAGE (6%
gel) of lysates of cells, transfected as indicated, were probed with
anti-(pRas-GRF1) immunoglobulin (upper panel) and anti-HA-probe
immunoglobulin (lower panel), to confirm equal loading. (B) Western
blots of SDS ⁄ PAGE (10% gel) of lysates of cells, transfected as
indicated, were probed with anti-pERK1 ⁄ 2 immunoglobulin (upper
panel) and anti-ERK1 ⁄ 2 immunoglobulin (lower panel), to confirm
equal loading.
Fig. 5. Serotonin increases intracellular Ca
2+
concentration through 5-hydroxytryptamine
7(b)
(5-HT
7(b)
) receptors. Non-transfected or stably
transfected human embryonic kidney (HEK)293 cells expressing the 5-HT
7(b)
receptor, KB1 cells, were cultured, washed and loaded with
5 l
M FURA-2-AM for 20 min. The fluorescence intensity in single cells was recorded at 340 nm and 380 nm for up to 300 s on an inverted
microscope. The cells were treated with 10 l
M serotonin 30 s subsequent to the start of the recordings, as indicated with an arrow. Inset,
in addition to treatment with FURA-2-AM, as described above, the cells were treated with carboxyamido-triazole (CAI) (20 l

M) or vehicle con-
trol (dimethylsulfoxide) for 25 min prior to treatment with 10 l
M serotonin.
J. H. Norum et al. Ras-GRF1 and Ras-dependent ERK activation in HEK293
FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS 2309
ment with 10 lm serotonin. Serotonin-induced phos-
phorylation of ERK1 ⁄ 2 was markedly reduced in the
presence of 20 lm CAI (Fig. 6A). Serotonin-induced
phosphorylation of ERK1 ⁄ 2 was also reduced in
cells incubated with the Ca
2+
chelator, BAPTA-AM
(40 lm), for 25 min prior to 5 min of treatment with
10 lm serotonin (Fig. 6B). Increasing the free intracel-
lular levels of Ca
2+
by treatment of HEK293 cells with
thapsigargin induced phosphorylation of ERK1 ⁄ 2
(Fig. 6C). Previously, CAI has been shown to inhibit
the thapsigargin-induced activation of ERK1 ⁄ 2in
Rat1 cells [29]. Thapsigargin-induced phosphorylation
of ERK1 ⁄ 2 in HEK293 cells was inhibited by pretreat-
ment with 20 lm CAI for 25 min, demonstrating that
CAI inhibited the calcium-mediated phosphorylation
of ERK1 ⁄ 2 under these conditions (Fig. 6C).
To determine whether the effect of CAI on the sero-
tonin-induced ERK1 ⁄ 2 phosphorylation was specific,
HEK293 cells were treated with 20 lm CAI for 25 min
prior to treatment with 10 nm EGF for 5 min. EGF-
induced phosphorylation of ERK1 ⁄ 2 was not influ-

enced by the presence of CAI (Fig. 6D), demonstrating
that CAI does not have a general suppressive effect on
the Ras-dependent activation of ERK1 ⁄ 2.
Increased basal ERK1 ⁄ 2 phosphorylation in the
presence of HA-Ras-GRF1 is reduced by CAI and
RasN17
In HEK293 cells transfected with the 5-HT
7(a)
recep-
tor, cotransfection with HA-Ras-GRF1 increased basal
ERK1 ⁄ 2 phosphorylation (Fig. 7A, lanes 5 and 6 vs.
lane 1). Serotonin-induced ERK1 ⁄ 2 phosphorylation
in these cotransfected cells was abolished by pretreat-
ment with CAI (Fig. 7A, lanes 5–12), as in cells trans-
A
B
C
D
Fig. 6. Serotonin-induced extracellular signal-regulated kinase
(ERK)1 ⁄ 2 phosphorylation is dependent on Ca
2+
. (A) Human embry-
onic kidney (HEK)293 cells transiently transfected with the 5-hy-
droxytryptamine
7(a)
(5-HT
7(a)
) receptor were treated with or without
20 l
M carboxyamido-triazole (CAI) for 25 min prior to treatment

with or without 10 l
M serotonin for 5 min, as indicated. (B)
HEK293 cells, transiently transfected with the 5-HT
7(a)
receptor,
were treated with or without 40 l
M BAPTA-AM for 25 min prior to
incubation for 5 min with or without 10 l
M serotonin. (C) and (D)
HEK293 cells were treated with or without 1 l
M thapsigargin (C) or
10 n
M epidermal growth factor (EGF) (D) for 5 min subsequent to
treatment with or without 20 l
M CAI for 25 min, as indicated. (A),
(B), (C) and (D) show representative western blots of proteins sep-
arated by SDS ⁄ PAGE (10% gel) and electroblotted over to
poly(vinylidene difluoride) membranes before probing with antibod-
ies, as indicated.
A
B
C
Fig. 7. Phosphorylation of extracellular signal-regulated kinase
(ERK)1 ⁄ 2, induced by recombinant HA-Ras-GRF1, is dependent on
Ca
2+
and Ras. (A) Human embryonic kidney (HEK)293 cells cotrans-
fected with the 5-hydroxytryptamine
7(a)
(5-HT

7(a)
) receptor and
empty vector or HA-Ras-GRF1 were treated with or without 10 l
M
serotonin for 5 min subsequent to treatment with 20 lM carbox-
yamido-triazole (CAI) or vehicle for 25 min, as indicated. (B) HEK293
cells transiently cotransfected with the 5-HT
7(a)
receptor and
HA-Ras-GRF1 were treated with or without 20 l
M CAI for 25 min
prior to treatment with or without 10 l
M serotonin for 5 min. (C)
HEK293 cells were cotransfected with 5-HT
7(a)
receptor and empty
vector, HA-Ras-GRF1 or RasN17, as indicated. The transfected cells
were treated with or without 10 l
M serotonin for 5 min. (A), (B) and
(C) show representative western blots of 10% (A and C) and 6%
(B) SDS ⁄ PAGE, probed with antibodies as indicated.
Ras-GRF1 and Ras-dependent ERK activation in HEK293 J. H. Norum et al.
2310 FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS
fected with the 5-HT
7(a)
receptor alone (Figs 6A and
7A). These results indicate that the serotonin-stimula-
ted ERK1 ⁄ 2 phosphorylation is Ca
2+
dependent.

There was also a slight inhibitory effect of CAI on the
increased basal phosphorylation of ERK1 ⁄ 2 observed
upon cotransfection with HA-Ras-GRF1 (Fig. 7A).
On the other hand, the serotonin-induced phosphory-
lation of HA-Ras-GRF1 was not affected by CAI,
indicating that this phosphorylation is not Ca
2+
dependent (Fig. 7B).
To determine whether the increased ERK1 ⁄ 2 phos-
phorylation in cells transfected with HA-Ras-GRF1
was mediated through Ras, HEK293 cells were
cotransfected with plasmids encoding the 5-HT
7(a)
receptor, HA-Ras-GRF1 and a dominant-negative
construct of Ras, RasN17. RasN17 essentially elimin-
ated the increase in ERK1 ⁄ 2 phosphorylation (both
basal and serotonin-stimulated) induced by the overex-
pression of HA-Ras-GRF1 (Fig. 7C), indicating that
the effect of Ras-GRF1 on basal and serotonin-stimu-
lated ERK1 ⁄ 2 phosphorylation is Ras-dependent.
Discussion
We report the endogenous expression of several
isoforms of the guanine nucleotide exchange factor
Ras-GRF1 in HEK293 cells. Serotonin treatment of
HEK293 cells, transiently transfected with the G
s
-cou-
pled 5-HT
7
receptors, induced cAMP ⁄ PKA-dependent

phosphorylation of endogenous Ras-GRF1 at Ser927
and recombinant mouse HA-tagged Ras-GRF1 at
Ser916. However, mutation of the Ser916 PKA phos-
phorylation site did not alter the increased basal or
serotonin-induced ERK1 ⁄ 2 phosphorylation induced
by the overexpression of HA-Ras-GRF1. A truncated
version of Ras-GRF1, lacking the Ca
2+
⁄ CaM-binding
IQ domain, did not increase the basal or serotonin-
induced ERK1 ⁄ 2 phosphorylation. The ERK1 ⁄ 2 phos-
phorylation was inhibited in the presence of the
calcium influx inhibitor, CAI.
The endogenous expression of 5-HT
6
and 5-HT
7
receptors has been reported in some HEK293 cells
[30]. However, in the current study, serotonin treat-
ment of nontransfected HEK293 cells did not result in
ERK1 ⁄ 2 phosphorylation or increased [Ca
2+
]
i
(data
not shown), indicating that the HEK293 cells used did
not show endogenous expression of functional 5-HT
7
or other G
s

-coupled serotonin receptors.
Ras-GRF1 contains several protein motifs that are
presumably involved in numerous regulatory mecha-
nisms. Binding of Ca
2+
⁄ CaM to the N-terminal IQ
motif is considered to be the main mechanism for
Ras-GRF1 activation [10]. Upon stimulation of
GPCRs Ras-GRF1 becomes phosphorylated on several
sites, with incompletely understood effects. The Ser916
residue of mouse Ras-GRF1 becomes phosphorylated
by PKA in vivo and in vitro [12]. This phosphorylation
is insufficient for activation but may enhance the activ-
ity of Ras-GRF1 towards Ras [12,14]. The phospho-
specific antibody that selectively recognizes mouse
and rat Ras-GRF1, which are phosphorylated at
Ser916 ⁄ 898, respectively, also recognizes human phos-
phorylated Ras-GRF1. The sequence surrounding
Ser927 in human Ras-GRF1 is homologous to that
surrounding Ser916 in mouse Ras-GRF1, with three
amino acid substitutions. In addition, several other
putative phosphorylation sites have been identified in
Ras-GRF1. Baouz and colleagues, for example, did
not find Ser916 as an in vitro PKA phosphorylation
site [13], but rather identified Ser745 and Ser822 as the
two most heavily phosphorylated residues. However,
compared with the human Ser927 sequence, the
sequences surrounding these two serine residues do not
align as well with the mouse Ser916 sequence. There-
fore, the phosphospecific antibody developed against

mouse phosphoSer916-Ras-GRF1 probably recognizes
human Ras-GRF1 phosphorylated at Ser927. The anti-
body is highly specific for the phosphorylated residue,
as mutations of Ser916 (in the mouse sequence) to
alanine, aspartic acid or glutamic acid were not recog-
nized by the antibody. Our finding, that reactivity of
the endogenous Ras-GRF1 in HEK293 cells to the
phospho-Ras-GRF1 antibody is stimulated by the acti-
vation of 5-HT
7
receptors, is also consistent with the
selective recognition of human Ras-GRF1 by this anti-
body when Ras-GRF1 is phosphorylated at Ser927.
The serotonin-induced phosphorylation of both
endogenous and recombinant Ras-GRF1 shows that
Ras-GRF1 is modified by stimulation with serotonin,
but is not direct evidence that Ras-GRF1 contributes
to the serotonin-induced activation of Ras and
ERK1 ⁄ 2. Pretreatment with H89 eliminated the sero-
tonin-induced phosphorylation of Ras-GRF1 at
Ser916 ⁄ 927. Transfection with the human phosphodi-
esterase PDE4D2 also reduced the serotonin-induced
Ras-GRF1 phosphorylation. In both cases, the sero-
tonin-induced ERK1 ⁄ 2 phosphorylation was lowered
concomitant with the reduced Ras-GRF1 phosphoryla-
tion, but ERK1 ⁄ 2 phosphorylation was only partially
reduced compared to the more substantial reduction of
Ras-GRF1 phosphorylation.
Neither preventing PKA-mediated phosphorylation
of mouse Ras-GRF1 Ser916 by mutating this residue

to alanine nor mutating the residue to either aspartic
or glutamic acid to potentially mimic the phosphoryla-
tion, influenced the increased basal or serotonin-
J. H. Norum et al. Ras-GRF1 and Ras-dependent ERK activation in HEK293
FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS 2311
induced ERK1 ⁄ 2 phosphorylation. Taken together,
these data indicate that the PKA-mediated phosphory-
lation of Ser916 of mouse Ras-GRF1, and presumably
Ser927 of human Ras-GRF1, does not have a central
role in ERK1 ⁄ 2 activation. The small differences in
Ras activation observed between wild-type Ras-GRF1
and the Ser916Ala mutant, both in vitro [12] and in an
assay of Ras-dependent neurite outgrowth from PC12
cells [14], may not be detectable at the level of
ERK1 ⁄ 2 phosphorylation owing to amplification of
the signal through the kinase cascade. These results are
also in agreement with our previous report that phos-
phorylation at this site was insufficient to activate
Ras-GRF1 in the absence of other signals [12]. It is
probable that phosphorylation at this site is only one
of several regulated phosphorylation events that occur
on Ras-GRF1 to regulate its activity in coordination
with increases in Ca
2+
, and so an effect from the
mutation of a single site may not be apparent. The
importance of phosphorylation of Ras-GRF1 at this
residue is underlined by the demonstration that it is a
physiologically relevant phosphorylation event which
occurs at the equivalent site (Ser898) in the dendritic

tree of rat prefrontal cortical neurones [14]. In addition
to regulation of the Ras GEF activity of Ras-GRF1,
other phosphorylation events, particularly on tyrosine
residues, may regulate its activity as a GEF for
another small G-protein, Rac [31].
Expression of recombinant, murine, HA-tagged Ras-
GRF1 (HA-Ras-GRF1) in HEK293 cells increased the
basal ERK1 ⁄ 2 phosphorylation compared to that of
nontransfected cells. Serotonin caused additional phos-
phorylation of ERK1 ⁄ 2 in HEK293 cells cotransfected
with the 5-HT
7(a)
receptor and HA-Ras-GRF1, but the
combined effect of 5-HT
7(a)
activation and HA-Ras-
GRF1 expression was not much higher than the sum
of the separate effects on ERK1 ⁄ 2 phosphorylation. If
endogenous Ras-GRF1 was the limiting factor in the
cascade from the 5-HT
7(a)
receptor to ERK1 ⁄ 2 phos-
phorylation, one might expect that the overexpression
of HA-Ras-GRF1 would elicit greater effects than
observed on ERK1 ⁄ 2 phosphorylation. On the other
hand, if endogenous Ras-GRF1 was not the limiting
factor in the cascade, one could hypothesize that the
effect of Ras-GRF1 overexpression on ERK1 ⁄ 2
phosphorylation would be similar to the sum of the
receptor-induced effect and increased basal phosphory-

lation, mediated from overexpressed Ras-GRF1, poss-
ibly localized in different cellular compartments from
the receptor.
The increased ERK1 ⁄ 2 phosphorylation in the pres-
ence of HA-Ras-GRF1 was essentially eliminated in
the presence of dominant-negative Ras, RasN17, but
only slightly reduced by the Ca
2+
influx inhibitor,
CAI. Both interventions prevented the serotonin-
induced phosphorylation of ERK1 ⁄ 2. A truncated ver-
sion of Ras-GRF1 (Ras-GRF1-D1-225) lacking the
PH1-, coiled-coil and IQ domain and thus not expec-
ted to bind Ca
2+
⁄ CaM, did not increase the basal
or serotonin-induced ERK1 ⁄ 2 phosphorylation. The
reduced ability of Ras-GRF1-D1-225 to induce
ERK1 ⁄ 2 activation may be a result of the lost
Ca
2+
⁄ CaM-binding site of the IQ domain, but the
missing PH1- and coiled-coil domains may also change
the subcellular localization of this version of Ras-
GRF1. These domains have been shown to contribute
to the regulation of Ras GEF activity [32]. The sero-
tonin-induced phosphorylation of Ras-GRF1-D1-225
at Ser916 indicates that the protein is located in cellu-
lar compartments within the reach of kinases activated
upon serotonin treatment. We have previously shown

that while increased intracellular Ca
2+
is required for
the stimulation of Ras-GRF1 activation by a G
i
-cou-
pled pathway [12], Ca
2+
does not stimulate Ras-GRF1
phosphorylation at Ser916 [14]. It is probable that
Ras-GRF1 can serve to integrate signals from the
cAMP and Ca
2+
second messenger cascades to deter-
mine activation of the ERK1 ⁄ 2 cascade. In addition to
the influence of second messengers and phosphoryla-
tion events on its activities, Ras-GRF1 can also be
regulated by interaction with another small GTPase,
Cdc42 [33], and can serve a scaffolding function that
directs signalling downstream of Ras activation [34,35].
In rat adrenal glomerulosa cells, 5-HT
7
receptors
were shown to increase [Ca
2+
]
i
through T-type Ca
2+
channels in a cAMP ⁄ PKA-dependent manner [22,23].

Increase in [Ca
2+
]
i
following stimulation of over-
expressed 5-HT
7(a)
receptors in HEK293 cells has pre-
viously been shown [36] and no evidence of coupling to
G
q
or G
i
was found. We showed that serotonin stimu-
lation of HEK293 cells stably expressing 5-HT
7(b)
receptors resulted in increased [Ca
2+
]
i
. Serotonin-
induced ERK1 ⁄ 2 phosphorylation was severely
reduced in the presence of CAI, but the PKA-depend-
ent phosphorylation of HA-Ras-GRF1 was not influ-
enced by the presence of CAI. In nonexcitable cells,
CAI can specifically inhibit store-operated calcium
channels and may thereby reduce the serotonin-
induced sustained increase in [Ca
2+
]

i
, as has been
shown for endothelin-1-induced Ca
2+
increase in Rat1
cells [29]. Whether HEK293 cells express T-type Ca
2+
channels, or whether the increase in [Ca
2+
]
i
is mediated
through a different mechanism, has not been addressed
further in this study, and the results obtained with the
calcium influx inhibitor, CAI, do not provide conclu-
sive data concerning the nature of the calcium increase.
Ras-GRF1 and Ras-dependent ERK activation in HEK293 J. H. Norum et al.
2312 FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS
Ras-GRF1 is implicated in signalling from various
neurotransmitter receptors [9,12,37]. The downstream
target of Ras-GRF1, Ras, may help to regulate expres-
sion of specific genes involved in processes such as
memory. In Aplysia, the activation of MAP kinases by
G
s
-coupled serotonin receptors is implicated in mem-
ory formation [38,39]. There is increasing evidence for
the biological importance of the Ras ⁄ MAP kinase cas-
cade in human learning and memory [40]. G
s

-coupled
serotonin receptors are found in the hippocampus
[41,42], and 5-HT
7
receptors activate ERK1 ⁄ 2 in cul-
tured neurones [43]. Ras-GRF1 is highly expressed in
hippocampal and other neurones, and Ras-GRF1-defi-
cient mice have memory defects [44,45]. Therefore, a
possible involvement of Ras-GRF1 in the Ca
2+
- and
Ras-dependent activation of ERK1 ⁄ 2 through 5-HT
7
receptors may be of physiological relevance. Since the
original manuscript was submitted for publication,
Johnson-Farley and colleagues have shown interplay
between G
s
- and G
q
-coupled serotonin receptors in the
activation of ERK1 ⁄ 2 and PKB (Akt) in PC12 cells
[46]. They found that PKA activation through G
s
-cou-
pled serotonin receptors was Ca
2+
dependent, whereas
ERK1 ⁄ 2 phosphorylation was Ca
2+

independent.
Considering all the different pathways reported for the
activation of Ras and ERK1 ⁄ 2 downstream of
GPCRs, Ras-GRF1 could be one of possibly several
GEFs involved in the activation of Ras and subse-
quently ERK1 ⁄ 2 downstream of G
s
-coupled serotonin
receptors. This remains a challenge for future research.
Experimental procedures
Materials
HEK293 cells were from the American Type Culture Collec-
tion (Manassas, VA, USA). Mouse monoclonal antiphos-
pho-ERK1 ⁄ 2 and rabbit polyclonal anti-(phosphoSer916-
Ras-GRF1) Ig (#3321) were from Cell Signaling Technology
(Beverly, MA, USA), sheep polyclonal antimouse immuno-
globulin–horseradish peroxidase conjugate (Ig-HRP) and
sheep anti-(rabbit IgG)–HRP were from Amersham Pharma-
cia Biotech (Little Chalfont, Bucks, UK), rabbit polyclonal
anti-ERK1 ⁄ 2 Ig was from Upstate Biotechnology (Lake
Placid, NY, USA), and rabbit polyclonal anti-(Ras-GRF1)
Ig (human, rat) was from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). 5-HT, EGF, H89 and Dulbecco’s modified
Eagle’s medium (DMEM) were from Sigma (St Louis, MO,
USA). Hybond-P [poly(vinylidene difluoride)] membrane
was from Amersham. Lipofectamine
TM
2000 was from Invi-
trogen (Carlsbad, CA, USA). Fetal bovine serum was from
EuroClone (Milano, Italy). UltraCULTURE

TM
general pur-
pose serum-free medium, penicillin ⁄ streptomycin and l-glu-
tamine were from Cambrex (Vervierse, Belgium). Supersignal
West Dura extended-duration chemiluminescent substrate
was from Pierce Biotechnology (Rockford, IL, USA), and
the BC assay protein quantification kit was from Uptima
(Monticon, France). BAPTA-AM was from Calbiochem (La
Jolla, CA, USA).
Plasmids
The pcDNA3.1(–) vector (Invitrogen), encoding the human
5-HT
7(a)
receptor, was as described previously [27]. The
pKH3 mammalian expression plasmids encoding the full-
length murine wild-type HA-Ras-GRF1 and the Ser916Ala
mutant were as described previously [9,12,47]. HA-Ras-
GRF1-Ser916Asp, HA-Ras-GRF1-Ser916Glu and HA-Ras-
GRF1-D1-225 were constructed by PCR using appropriate
mutagenic primers and the protocol previously described
[12] and then confirmed by DNA sequencing. The pCMV
vector encoding dominant-negative Ras, RasN17, was from
Clontech (Palo Alto, CA, USA). The pCMV5 vector enco-
ding the human phosphodiesterase 4D2, hPDE4D2, was
provided by M. Conti (Department of Obstetrics and
Gynaecology, Stanford, CA, USA).
Cell culture and transfection
HEK293 cells were cultured in DMEM containing 10%
(v ⁄ v) fetal bovine serum and supplements (2 mml-gluta-
mine, 100 UÆmL

)1
penicillin, 100 lg ÆmL
)1
streptomycin), at
37 °C in a humidified atmosphere of 5% CO
2
in air, and
transfected at 60–70% confluence with the indicated
cDNA(s) using Lipofectamine 2000, according to the manu-
facturer’s protocol. When necessary, empty vector
[pcDNA3.1(–)] was included in the transfection to ensure
that each dish received the same amount of DNA (1.0 or
2.9 lg of plasmid DNA per 35 or 60 mm dish, respectively).
Cells expressing 5-HT
7
receptors were cultured in UltraCUL-
TURE
TM
serum-free medium with supplements, as described
above, prior to starvation in DMEM without serum for the
last 16–20 h before serotonin treatment and lysis ( 48 h
after transfection for transiently transfected cells). Non-
transfected cells were similarly starved in DMEM without
serum before treatment (with EGF or thapsigargin) and lysis.
Where indicated, cells were preincubated with 20 lm H89,
20 lm CAI or 40 lm BAPTA-AM for 25 min prior to treat-
ment with agonist. Cells were stimulated for 5 min if not
indicated otherwise. All experiments were carried out in
duplicate at least three times, if not otherwise indicated.
Western Blotting

Equal amounts of cell lysate proteins were separated by
SDS ⁄ PAGE and electroblotted onto poly(vinylidene difluo-
ride) membranes. The membranes were incubated with pri-
J. H. Norum et al. Ras-GRF1 and Ras-dependent ERK activation in HEK293
FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS 2313
mary antibodies [anti-(phospho-ERK1 ⁄ 2), 1 : 2000, v ⁄ v;
anti-ERK1 ⁄ 2, 1 : 10 000, v ⁄ v; anti-(phospho-Ras-GRF1),
1 : 1000, v ⁄ v; and anti-(Ras-GRF1), 1 : 1000, v ⁄ v] in
NaCl ⁄ P
i
containing 5% (w⁄ v) nonfat dry milk and 0.05%
(v ⁄ v) Tween 20, and thereafter incubated with the corres-
ponding HRP-conjugated secondary antibodies. The
immobilized HRP-conjugated secondary antibodies were
visualized with SuperSignal Dura West extended-duration
chemiluminescent substrate and analysed with a UVP
BioChemie system.
Phospho-ERK1 ⁄ 2 and phospho-Ras-GRF1 assay
The cells were cultured in 35 mm dishes, transfected and
stimulated with agonist as described, lysed in ice-cold cell
lysis buffer [1% (w ⁄ v) SDS, 1 mm Na
3
VO
4
,50mm
Tris ⁄ HCl, pH 7.4, at room temperature], scraped with a
Teflon cell scraper, sheared through a 25 GA syringe and
immediately frozen in liquid nitrogen. The thawed cell ly-
sates were cleared by centrifugation (13 000 g at 4 °C) and
the protein concentrations in the supernatants were quanti-

fied by the BC assay quantification kit (Uptima) using BSA
as the standard. Equal amounts of protein were prepared
for separation by SDS ⁄ PAGE.
Immunoprecipitation
HEK293 cells were cultured in 60 mm dishes and grown as
described above, lysed in ice-cold lysis buffer and the lysate
cleared by centrifugation (13 000 g at 4 °C). Samples con-
taining 200 lg of protein were diluted in ice-cold 1 · IP buf-
fer [10 mm Tris ⁄ HCl, pH 7.4 at room temperature, 150 mm
NaCl, 1 mm EDTA, 1 mm EGTA, 0.2 mm Na
3
VO
4
, 0.2 mm
phenylmethanesulfonyl fluoride, 1% (v ⁄ v) Triton X-100,
0.5% (v ⁄ v) Nonidet P-40) to a final volume of 1 mL and
incubated for 2 h at 4 °C with or without (control) poly-
clonal anti-Ras-GRF1 immunoglobulin and subsequently
with sheep anti-rabbit paramagnetic beads (Dynal, Oslo,
Norway) overnight at 4 °C on a tumbler. The beads with
the bound proteins were washed three times with cold
1 · IP buffer. The proteins were eluted from the beads by
boiling for 5 min in 1· SDS ⁄ PAGE loading buffer [31 mm
Tris ⁄ HCl, pH 6.8 at room temperature, 1% (w ⁄ v) SDS,
2.5% (v ⁄ v) glycerol, 0.025% (w ⁄ v) bromophenol blue, 2.5%
(v ⁄ v) b-mercaptoethanol]. The protein samples were loaded
and resolved on 6% (w ⁄ v) SDS ⁄ polyacrylamide gels.
Western blotting was performed as described above.
Isolation of mRNA, and PCR
Total RNA from HEK293 cells, cultured as described

above, was isolated by using the SV total RNA isolation
system (Promega, Madison, WI, USA), according to the
manufacturer’s protocol. Paramagnetic beads were used to
isolate mRNA from the pool of total RNA. The purified
mRNA was used for oligo-dT primed cDNA synthesis. The
cDNA was treated with RNase H to remove RNA comple-
mentary to the cDNA. The purified cDNA was used as
substrate in PCR with the following Ras-GRF1 gene-speci-
fic primers: ON357, 5¢-TGAAACATCACCAACTAAATC
TCCAA-3¢; ON358, 5¢-GACGACTCCATTGTTATAGG
AAAAGAGT-3¢; ON359, 5 ¢-GCCGCTGGAGAAACAG
CAT-3¢; ON360, 5¢-GCCACCCATTCGTCACAATC-3¢;
and ON361, 5¢-ATGCAGAAGGGGATCCGG-3¢.
Direct measurements of [Ca
2+
]
i
in single cells
Non-transfected HEK293 cells and HEK293 cells stably
transfected with the 5-HT
7(b)
receptor (KB1 cells) were
plated and cultured in specially designed glass-bottomed
wells [48] coated with 12.7 lgÆcm
)2
collagen type VII from
rat tail. At 70–80% confluence, the cells were washed at
37 °C with Hepes-buffered salt solution (HSS; 136 mm
NaCl, 5 mm KCl, 1.2 mm MgCl
2

, 1.2 mm CaCl
2
,11mm
Bacto-dextrose, 10 mm Hepes, pH 7.35) and incubated
with 5 lm FURA-2-AM in HSS for 20 min at room tem-
perature. The cells were washed once at 37 °C with HSS
buffer prior to mounting the cell culture dish on an
inverted Nikon microscope equipped with digital record-
ing facilities. Recordings of the fluorescence intensities
started 30 s prior to the addition of vehicle or serotonin
solution. The fluorescence intensity of FURA-2 at excita-
tion wavelength 340 nm (F340) increases, whereas the
fluorescence intensity at 380 nm (F380) decreases upon
the binding of Ca
2+
. The change in the ratio of
F340 ⁄ F380 determined the change in Ca
2+
concentration
inside the cell.
Acknowledgements
This work was supported by The Norwegian Council on
Cardiovascular Diseases, The Norwegian Cancer Soci-
ety, The Research Council of Norway, The Novo Nor-
disk Foundation, Anders Jahre’s Foundation for the
Promotion of Science, The Blix Family Foundation and
grants from the University of Oslo and the National
Cancer Institute of the USA (R01-CA81150). The
experiments were performed in accordance with all regu-
lations concerning biomedical research in Norway. We

thank Dr Marco Conti for generating and providing the
plasmid encoding hPDE4D2 and thank Dr Jens-Gustav
Iversen for help with the calcium measurements.
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