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

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

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-

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

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

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

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

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

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)

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

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

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

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

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.

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

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

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-

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].

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

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

)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

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

, 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

5 Dikic I, Tokiwa G, Lev S, Courtneidge SA & Schles-
singer J (1996) A role for Pyk2 and Src in linking G-
protein-coupled receptors with MAP kinase activation.
Nature 383, 547–550.
6 Toki S, Kawasaki H, Tashiro N, Housman DE &
Graybiel AM (2001) Guanine nucleotide exchange fac-
tors CalDAG-GEFI and CalDAG-GEFII are coloca-
lized in striatal projection neurons. J Comp Neurol 437,
398–407.
7 Wolfman A & Macara IG (1990) A cytosolic protein
catalyzes the release of GDP from p21ras. Science 248,
67–69.
8 Shou C, Wurmser A, Ling K, Barbacid M & Feig LA
(1995) Differential response of the Ras exchange factor,
Ras-GRF to tyrosine kinase and G-protein mediated
signals. Oncogene 10, 1887–1893.
9 Mattingly RR & Macara IG (1996) Phosphorylation-
dependent activation of the Ras-GRF ⁄ CDC25
Mm
exchange factor by muscarinic receptors and G-protein
bc subunits. Nature 382, 268–272.
10 Farnsworth CL, Freshney NW, Rosen LB, Ghosh A,
Greenberg ME & Feig LA (1995) Calcium activation of
Ras mediated by neuronal exchange factor Ras-GRF.
Nature 376, 524–527.
11 Mattingly RR, Saini V & Macara IG (1999) Activation
of the Ras-GRF ⁄ CDC25
Mm
exchange factor by lyso-
phosphatidic acid. Cell Signal 11, 603–610.

18 Guerrero C, Rojas JM, Chedid M, Esteban LM,
Zimonjic DB, Popescu NC, deMora JF & Santos E
(1996) Expression of alternative forms of Ras exchange
factors GRF and SOS1 in different human tissues and
cell lines. Oncogene 12, 1097–1107.
19 Martegani E, Vanoni M, Zippel R, Coccetti P,
Brambilla R, Ferrari C, Sturani E & Alberghina L
(1992) Cloning by functional complementation of a
mouse cDNA encoding a homologue of CDC25, a
Saccharomyces cerevisiae RAS activator. EMBO J 11,
2151–2157.
20 Heidmann DE, Metcalf MA, Kohen R & Hamblin MW
(1997) Four 5-hydroxytryptamine
7
(5-HT
7
) receptor iso-
forms in human and rat produced by alternative spli-
cing: species differences due to altered intron-exon
organization. J Neurochem 68, 1372–1381.
21 de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH,
Nijman SM, Wittinghofer A & Bos JL (1998) Epac is a
Rap1 guanine-nucleotide-exchange factor directly acti-
vated by cyclic AMP. Nature 396, 474–477.
22 Lenglet S, Louiset E, Delarue C, Vaudry H & Contesse
V (2002) Involvement of T-type calcium channels in the
mechanism of action of 5-HT in rat glomerulosa cells: a
novel signaling pathway for the 5-HT
7
receptor. Endocr

signaling is required for normal beta-cell development
and glucose homeostasis. EMBO J 22, 3039–3049.
27 Krobert KA, Bach T, Syversveen T, Kvingedal AM &
Levy FO (2001) The cloned human 5-HT
7
receptor
splice variants: a comparative characterization of their
pharmacology, function and distribution. Naunyn-
Schmiedeberg’s Arch Pharmacol 363, 620–632.
28 Davies SP, Reddy H, Caivano M & Cohen P (2000)
Specificity and mechanism of action of some com-
monly used protein kinase inhibitors. Biochem J 351,
95–105.
29 Rodland KD, Wersto RP, Hobson S & Kohn EC
(1997) Thapsigargin-induced gene expression in nonexci-
table cells is dependent on calcium influx. Mol Endocri-
nol 11, 281–291.
30 Johnson MS, Lutz EM, Firbank S, Holland PJ &
Mitchell R (2003) Functional interactions between
native G
s
-coupled 5-HT receptors in HEK-293 cells and
the heterologously expressed serotonin transporter. Cell
Signal 15, 803–811.
31 Kiyono M, Kaziro Y & Satoh T (2000) Induction of
Rac-guanine nucleotide exchange activity of Ras-
GRF1 ⁄ CDC25
Mm
following phosphorylation by the
nonreceptor tyrosine kinase Src. J Biol Chem 275, 5441–

7A
receptor. J Biol
Chem 273, 17469–17476.
37 Kiyono M, Satoh T & Kaziro Y (1999) G protein bc
subunit-dependent Rac-guanine nucleotide exchange
activity of Ras-GRF1 ⁄ CDC25
Mm
. Proc Natl Acad Sci
USA 96, 4826–4831.
38 Martin KC, Michael D, Rose JC, Barad M, Casadio A,
Zhu H & Kandel ER (1997) MAP kinase translocates
into the nucleus of the presynaptic cell and is required for
long-term facilitation in Aplysia. Neuron 18, 899–912.
39 Michael D, Martin KC, Seger R, Ning MM, Baston R
& Kandel ER (1998) Repeated pulses of serotonin
required for long-term facilitation activate mitogen-
activated protein kinase in sensory neurons of Aplysia.
Proc Natl Acad Sci USA 95, 1864–1869.
40 Weeber EJ, Levenson JM & Sweatt JD (2002) Molecu-
lar genetics of human cognition. Mol Interv 2, 376–391.
41 Markstein R, Matsumoto M, Kohler C, Togashi H,
Yoshioka M & Hoyer D (1999) Pharmacological char-
acterisation of 5-HT receptors positively coupled to ade-
nylyl cyclase in the rat hippocampus. Naunyn-
Schmiedeberg’s Arch Pharmacol 359, 454–459.
42 Thomas DR, Middlemiss DN, Taylor SG, Nelson P &
Brown AM (1999) 5-CT stimulation of adenylyl cyclase
activity in guinea-pig hippocampus: evidence for invol-
vement of 5-HT
7

by the Ras-GTPase-activating protein SH3 domain. Mol
Cell Biol 14, 7943–7952.
48 Røtnes JS & Iversen JG (1992) Thapsigargin reveals
evidence for fMLP-insensitive calcium pools in human
leukocytes. Cell Calcium 13, 487–500.
Ras-GRF1 and Ras-dependent ERK activation in HEK293 J. H. Norum et al.
2316 FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS