GIPC (GAIP interacting protein, C terminus) is a PDZ-domain-
containing protein initially identified by virtue of its interaction with
RGS-GAIP (regulator of G protein signaling-GTPase activating
protein for G?i) (De Vries et al., 1998b). The list of identified
binding partners for GIPC includes numerous membrane proteins
(Katoh, 2002), such as the transmembrane semaphorin M-SemF
(Wang et al., 1999), neuropilin 1 (Cai and Reed, 1999), the Wnt
receptor frizzled 3 (Tan et al., 2001), the ?1-adrenergic receptor (Hu
et al., 2003), the TGF?-type III receptor (Blobe et al., 2001),
melanosomal membrane protein gp75 (tyrosinase-related protein 1)
(Liu et al., 2001), the tyrosine kinase receptor TrkA (Lou et al.,
2001), intergrin ? subunits (El Mourabit et al., 2002; Tani and
Mercurio, 2001), insulin-like growth factor 1 receptor (IGF1R)
(Booth et al., 2002; Ligensa et al., 2001), human lutropin receptor
(Hirakawa et al., 2003), myosin VI (Dance et al., 2004; Hasson,
2003), dopamine D2 and D3 receptors (Jeanneteau et al., 2004a;
Jeanneteau et al., 2004b), a myeloid cell-surface marker CD93
(Bohlson et al., 2005) and human papillomavirus type 18 E6 protein
(Favre-Bonvin et al., 2005). Although the list of GIPC-binding
partners is long, the endogenous function of GIPC and the
physiological significance of these associations are much less
studied. Loss-of-function studies published so far have been
primarily in ex vivo or in vitro systems, such as Xenopusectodermal
explants (Tan et al., 2001) and cultured cell lines (Favre-Bonvin et
al., 2005; Hirakawa et al., 2003); the in vivo role of GIPC in
embryonic development has not yet been examined.
Previously, we identified a Xenopus homolog of GIPC, kermit
(Tan et al., 2001), that is 72% identical to mammalian GIPC (De
Vries et al., 1998b). Knockdown of kermit using antisense
morpholino oligonucleotides blocked neural crest induction by
Xenopus frizzled 3 in ectodermal (animal cap) explants (Tan et al.,
2001), but did not inhibit neural crest formation in whole embryos.
We reasoned that this negative result may indicate the presence of a
compensating or redundant activity in whole embryos that is absent
in animal cap explants. We therefore identified a second kermitgene
(67% amino acid identity with kermit 1) in Xenopus early embryos
to study the redundancy between kermitand kermit2. Unexpectedly,
we discovered a novel function for kermit 2 in IGF signaling that
does not overlap with kermit 1.
Kermit 2 is identical to XGIPC, identified in a yeast two hybrid
screen for IGF1 receptor binding proteins in Xenopus oocytes
(Booth et al., 2002). XGIPC/kermit 2 binds to the cytoplasmic
domain of the XenopusIGF1R and this interaction appears to require
the PDZ domain of XGIPC. Overexpression of C-terminal
truncation mutants of XGIPC that retain the PDZ domain blocks
insulin-induced Xenopus MAP kinase activation and oocyte
maturation. Human GIPC was also identified as a binding partner
for human IGF1R (Ligensa et al., 2001).
IGF signaling has been recently implicated in neural induction
in Xenopus and zebrafish embryos (Eivers et al., 2004; Pera et al.,
2003; Pera et al., 2001; Richard-Parpaillon et al., 2002). Ectopic
expression of IGF in dorsal cells leads to the induction of ectopic
eyes and eye expansion and, when expressed in ventral cells,
induces secondary head-like structures (Pera et al., 2001; Richard-
Parpaillon et al., 2002). Inhibition of IGF signaling by either
dominant-negative IGF1R or IGF1R depletion reduces head
structures (Pera et al., 2001; Richard-Parpaillon et al., 2002). As
kermit 2/XGIPC physically interacts with XIGF1R, we examined
the involvement of kermit 2/XGIPC in IGF signaling in Xenopus
We report here that kermit2/XGIPC is expressed throughout
Xenopus early embryonic development and is localized to the
anterior region during neurula stage in a pattern highly similar to
XIGF1R (Richard-Parpaillon et al., 2002). Knockdown of kermit 2
leads to embryos with reduced anterior structures, specifically
reduction in the presumptive eye region. Furthermore, depletion
of kermit 2 and expression of dominant-negative XIGF1R
Kermit 2/XGIPC, an IGF1 receptor interacting protein, is
required for IGF signaling in Xenopus eye development
Jinling Wu1, Michael O’Donnell2, Aaron D. Gitler1and Peter S. Klein1,2,*
GIPC is a PDZ-domain-containing protein identified in vertebrate and invertebrate organisms through its interaction with a variety
of binding partners including many membrane proteins. Despite the multiple reports identifying GIPC, its endogenous function and
the physiological significance of these interactions are much less studied. We have previously identified the Xenopus GIPC homolog
kermit as a frizzled 3 interacting protein that is required for frizzled 3 induction of neural crest in ectodermal explants. We
identified a second Xenopus GIPC homolog, named kermit 2 (also recently described as an IGF receptor interacting protein and
named XGIPC). Despite its high amino acid similarity with kermit, kermit 2/XGIPC has a distinct function in Xenopus embryos. Loss-
of-function analysis indicates that kermit 2/XGIPC is specifically required for Xenopus eye development. Kermit 2/XGIPC functions
downstream of IGF in eye formation and is required for maintaining IGF-induced AKT activation. A constitutively active PI3 kinase
partially rescues the Kermit 2/XGIPC loss-of-function phenotype. Our results provide the first in vivo loss of function analysis of GIPC
in embryonic development and also indicate that kermit 2/XGIPC is a novel component of the IGF pathway, potentially functioning
through modulation of the IGF1 receptor.
KEY WORDS: Kermit 2, GIPC, Insulin like growth factor (IGF), IGF1 receptor (IGF1R), Eye development, Xenopus
Development 133, 3651-3660 (2006) doi:10.1242/dev.02547
1Cell and Molecular Biology Graduate Group, University of Pennsylvania School of
Medicine, 364 Clinical Research Building, 415 Curie Blvd, Philadelphia, PA 19104,
USA. 2Department of Medicine (Hematology-Oncology), University of Pennsylvania
School of Medicine, 364 Clinical Research Building, 415 Curie Blvd, Philadelphia,
PA 19104, USA.
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 18 July 2006
synergistically inhibit eye development. Kermit 2 is required for
IGF1 induced eye formation in whole embryos and for the induction
of eye molecular markers in ectodermal explants. Finally, we present
evidence that kermit 2 is required to maintain IGF/PI3 kinase-
dependent activation of AKT. These results indicate that kermit
2/XGIPC is required for IGF signaling in Xenopuseye development
likely through its interaction with the IGF1 receptor.
MATERIALS AND METHODS
Cloning of kermit2, plasmid constructs and in vitro transcription
kermit2 was identified through a Blast search of the Xenopus EST database
using the kermit sequence (Tan et al., 2001). The GenBank accession
number for kermit2 is BC089139. kermit2 5?UTR sequence was identified
from a stage 10 Xenopus 5?RACE cDNA library. Capped mRNAs were
synthesized by in vitro transcription of plasmids encoding XIGF1R
(Richard-Parpaillon et al., 2002), DN-IGFR (Pera et al., 2001), XIGF1
(Pera et al., 2001), p110* (Carballada et al., 2001), kermit 2/pCS2, kermit
2-GFP/pCS2 and 5?UTR-kermit 2-GFP/pCS2 (described below) using SP6
mMessage mMachine kits (Ambion). Kermit 2/pCS2 was cloned by
RT-PCR amplifying stage 1 Xenopus RNA using primers (forward,
AGGGATCCATGCCTCTGGGATTGCGCGTA; reverse, AGTCTAGAT-
TAAAAGCGTCCTTGTTTAGC) with engineered BamHI and XbaI sites
and cloned directionally into pCS2+. Kermit 2-GFP/pCS2 was generated
by PCR using kermit 2/pCS2 as the template and cloned into
BamHI/EcoRI sites of pCS2-EGFP. 5?UTR-kermit 2-GFP/pCS2 was
cloned by PCR amplifying using primers (upstream, CGGGATC-
TTCAAAGCGTCCTTGTTTAGCATC) with engineered BamHI and
EcoRI sites and cloned directionally into pCS2-EGFP.
RNA isolation and RT-PCR methods are described elsewhere (Yang et al.,
2002). Primers for EF-1? (Yang et al., 2002), ODC (Yang et al., 2002),
Otx2 (Cox and Hemmati-Brivanlou, 1995), En2 (Hemmati-Brivanlou et
al., 1994), muscle actin (Hemmati-Brivanlou and Melton, 1994), XAG
(Blitz and Cho, 1995) and Xrx (Mathers et al., 1997) have been described
previously. Primers were designed to detect kermit2 (upstream, ATGC-
CTCTGGGATTGCGCGTAAAG; downstream, TTTAACTTTGTCA-
ATCGTGCTCTC), HoxD1 (upstream, CACTTCTTGCGGGGATGTTT;
downstream, AGAGTCCTGTAGCTCAGCTG), Pax6 (by N. Hirsch and
W. Harris; upstream, AGTGTCCTCATTCACATCG; downstream, AG-
TACTGAGACATGTCAGG) and Vent1 (upstream, GCATCTCCTTG-
GCATATTTGG; downstream, TTCCCTTCAGCATGGTTCAAC).
Lineage tracing and in situ hybridization
Embryos were co-injected with RNA encoding ?-galactosidase with a
nuclear localization motif and control morpholino or kermit 2 morpholino
(5?-AGAGGCATCTTTCTTTCAGCGAAGG-3?). ?-Galactosidase activity
was visualized in embryos with Red-Gal (Research organics). Whole-mount
in situ hybridization was performed as described (Deardorff et al., 1998).
Antisense probes detected chordin (Sasai et al., 1994), gooscoid (Blumberg
et al., 1991), Bf1 (Papalopulu and Kintner, 1996), Otx2 (Lamb et al., 1993),
Pax6 (Hirsch and Harris, 1997) and Xrx (Mathers et al., 1997). For sense
probes, kermit 2/pCS2 was linearized by NotI and transcribed by Sp6
polymerase. For antisense probes, kermit 2/pCS2 was linearized by BamHI
and transcribed by T7 polymerase.
Embryos, microinjection, immunoprecipitation and
Eggs were obtained from Xenopus females, in vitro fertilized and
microinjected as described (Deardorff et al., 1998). Each injected blastomere
received 10 nl of RNA or morpholino. For unilateral injections, morpholino
and/or mRNA were injected into one dorsal blastomere at the four-cell stage.
Animal pole explants assays were performed as described previously
(Deardorff et al., 2001). For immunoprecipitation, Xenopus embryos were
injected in the animal pole at the one-cell stage, cultured to stage 10 and
lysed in embryo lysis buffer [20 mM Tris (pH 7.5), 140 mM NaCl, 10%
glycerol, 1 mM DTT, 2 mM sodium vanadate, 25 mM NaF, 1% Nonidet P-
40 and protease inhibitor cocktail for mammalian cells (Sigma)]. Anti-GFP
monoclonal antibody (1 ?l) (Roche, 1 814 460) was incubated with 250 ?l
of cleared lysate overnight at 4°C, collected on 30 ?l protein G agarose
beads (Invitrogen, 15920-010) for 1 hour, washed three times with PBS, and
analyzed by western blot using IGF1R antibody (Cell Signaling, 3022).
AKT, P-AKT, MAPK, P-MAPK and ?-tubulin antibodies are from Sigma
(p2482), Cell Signaling (9271), Cell Signaling (9102), Sigma (M9692) and
BD Biosciences (556321), respectively.
TUNEL assay and phosphorylated histone H3 staining
Phosphorylated histone H3 staining was performed as described (Bellmeyer
et al., 2003). TUNEL staining protocol was from www.xenbase.org
Expression of kermit2/XGIPC in Xenopus embryos
To characterize the temporal pattern of kermit2 expression, we
extracted RNA from multiple stages of embryogenesis and
performed RT-PCR. kermit2 was detected maternally (four-cell
embryos) and throughout early embryonic development (Fig. 1A).
kermit2 is ubiquitously expressed in early and mid-neurula stage
embryos (Fig. 1B and data not shown). As neurulation progresses,
kermit2 expression is restricted to the anterior region (Fig. 1B,D),
including the cement gland and neural plate border, adjacent to
and/or overlapping the presumptive eye region (Fig. 1D). The
expression pattern of kermit2is very similar to XIGF1Rat this stage
(Richard-Parpaillon et al., 2002). By tailbud stages, kermit2
expression becomes further restricted, with strong expression in the
cement gland, otic vesicles (Fig. 1E), pronephros, branchial arches
(Fig. 1F), and cranial nerves V, VII and IX (data not shown).
Kermit2/XGIPC is required for eye development
To study the endogenous function of kermit 2/XGIPC in Xenopus
embryos, we depleted kermit 2 using morpholino antisense
oligonucleotides. The kermit 2 morpholino blocked translation of
kermit 2-GFPmRNA containing the endogenous 5?UTR but had no
effect on kermit 2-GFP mRNA lacking the 5?UTR (Fig. 2A).
Unilateral injection (into one dorsal blastomere at the four-cell stage)
of kermit 2 morpholino strongly inhibited anterior development,
especially eye development, in a dose-dependent manner (Fig. 2B;
data not shown). Although there was a distribution of phenotypes,
60% (n=38) of embryos receiving 40 ng of kermit 2 morpholino
developed without any detectable eyes on the injected side and 32%
had small eyes (Fig. 2B). Co-injection of kermit2-GFP mRNA
lacking the morpholino target sequence restored eye formation in
72% (n=43) of kermit2-depleted embryos, and of these, 28%
appeared completely normal (Fig. 2B). kermit2-GFP mRNA alone
did not cause apparent developmental defects. In addition, bilateral
injection of kermit 2 morpholino caused absent or miniscule eyes in
83% (n=73) of embryos, and this effect was reversed in 60% (n=85)
of embryos co-injected with the kermit2-GFP rescuing mRNA,
although bilateral injections were also associated with more severe
trunk defects that were not completely rescued by kermit2-GFP
(data not shown). These results suggest that the reduced eye
formation phenotype is specifically due to loss of kermit 2.
To characterize further the kermit 2 loss-of-function phenotype,
we analyzed molecular markers at various stages by in situ
hybridization. Kermit 2 depletion disrupted anterior development,
which could indicate direct inhibition of anterior neural development
or could alternatively indicate mild ventralization. To examine
whether kermit 2 plays a role in dorsal development, we examined
the expression of the dorsal organizer genes chordinand goosecoid.
Kermit 2 or control morpholinos were injected into two dorsal
animal blastomeres of four-cell/eight-cell embryos, and chordin or
Development 133 (18)
Hirsch, N. and Harris, W. A. (1997). Xenopus Pax-6 and retinal development. J.
Neurobiol. 32, 45-61.
Hu, L. A., Chen, W., Martin, N. P., Whalen, E. J., Premont, R. T. and
Lefkowitz, R. J. (2003). GIPC interacts with the beta1-adrenergic receptor and
regulates beta1-adrenergic receptor-mediated ERK activation. J. Biol. Chem. 278,
Jeanneteau, F., Diaz, J., Sokoloff, P. and Griffon, N. (2004a). Interactions of
GIPC with dopamine D2, D3 but not D4 receptors define a novel mode of
regulation of G protein-coupled receptors. Mol. Biol. Cell 15, 696-705.
Jeanneteau, F., Guillin, O., Diaz, J., Griffon, N. and Sokoloff, P. (2004b). GIPC
recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling. Mol. Biol.
Cell 15, 4926-4937.
Katoh, M. (2002). GIPC gene family (Review). Int. J. Mol. Med. 9, 585-589.
Kuroda, H., Fuentealba, L., Ikeda, A., Reversade, B. and De Robertis, E. M.
(2005). Default neural induction: neuralization of dissociated Xenopus cells is
mediated by Ras/MAPK activation. Genes Dev. 19, 1022-1027.
Lamb, T. M., Knecht, A. K., Smith, W. C., Stachel, S. E., Economides, A. N.,
Stahl, N., Yancopolous, G. D. and Harland, R. M. (1993). Neural Induction by
the Secreted Polypeptide Noggin. Science 262, 713-718.
Lefkowitz, R. J. and Shenoy, S. K. (2005). Transduction of receptor signals by
beta-arrestins. Science 308, 512-517.
Ligensa, T., Krauss, S., Demuth, D., Schumacher, R., Camonis, J., Jaques, G.
and Weidner, K. M. (2001). A PDZ domain protein interacts with the C-
terminal tail of the insulin-like growth factor-1 receptor but not with the insulin
receptor. J. Biol. Chem. 276, 33419-33427.
Lin, F. T., Daaka, Y. and Lefkowitz, R. J. (1998). beta-arrestins regulate
mitogenic signaling and clathrin-mediated endocytosis of the insulin-like growth
factor I receptor. J. Biol. Chem. 273, 31640-31643.
Liu, T. F., Kandala, G. and Setaluri, V. (2001). PDZ domain protein GIPC interacts
with the cytoplasmic tail of melanosomal membrane protein gp75 (tyrosinase-
related protein-1). J. Biol. Chem. 276, 35768-35777.
Lou, X., Yano, H., Lee, F., Chao, M. V. and Farquhar, M. G. (2001). GIPC and
GAIP form a complex with TrkA: a putative link between G protein and receptor
tyrosine kinase pathways. Mol. Biol. Cell 12, 615-627.
Lou, X., McQuistan, T., Orlando, R. A. and Farquhar, M. G. (2002). GAIP, GIPC
and Galphai3 are concentrated in endocytic compartments of proximal tubule
cells: putative role in regulating megalin’s function. J. Am. Soc. Nephrol. 13,
Luttrell, L. M., Roudabush, F. L., Choy, E. W., Miller, W. E., Field, M. E., Pierce,
K. L. and Lefkowitz, R. J. (2001). Activation and targeting of extracellular
signal-regulated kinases by beta-arrestin scaffolds. Proc. Natl. Acad. Sci. USA 98,
Mathers, P. H., Grinberg, A., Mahon, K. A. and Jamrich, M. (1997). The Rx
homeobox gene is essential for vertebrate eye development. Nature 387, 603-
O’Connor, R. (2003). Regulation of IGF-I receptor signaling in tumor cells. Horm.
Metab. Res. 35, 771-777.
Oldham, S. and Hafen, E. (2003). Insulin/IGF and target of rapamycin signaling: a
TOR de force in growth control. Trends Cell Biol. 13, 79-85.
Papalopulu, N. and Kintner, C. (1996). A posteriorising factor, retinoic acid,
reveals that anteroposterior patterning controls the timing of neuronal
differentiation in Xenopus neuroectoderm. Development 122, 3409-3418.
Payne, D. M., Rossomando, A. J., Martino, P., Erickson, A. K., Her, J. H.,
Shabanowitz, J., Hunt, D. F., Weber, M. J. and Sturgill, T. W. (1991).
Identification of the regulatory phosphorylation sites in pp42/mitogen-activated
protein kinase (MAP kinase). EMBO J. 10, 885-892.
Pera, E. M., Wessely, O., Li, S. Y. and De Robertis, E. M. (2001). Neural and
head induction by insulin-like growth factor signals. Dev. Cell 1, 655-665.
Pera, E. M., Ikeda, A., Eivers, E. and De Robertis, E. M. (2003). Integration of
IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction.
Genes Dev. 17, 3023-3028.
Richard-Parpaillon, L., Heligon, C., Chesnel, F., Boujard, D. and Philpott, A.
(2002). The IGF pathway regulates head formation by inhibiting Wnt signaling in
Xenopus. Dev. Biol. 244, 407-417.
Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. K. and De Robertis, E.
M. (1994). Xenopus chordin: a novel dorsalizing factor activated by organizer-
specific homeobox genes. Cell 79, 779-790.
Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G.
F., Holmes, A. B., Gaffney, P. R., Reese, C. B., McCormick, F., Tempst, P. et
al. (1998). Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-
trisphosphate-dependent activation of protein kinase B.[see comment]. Science
Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R., Reese, C. B., Painter,
G. F., Holmes, A. B., McCormick, F. and Hawkins, P. T. (1997). Dual role of
phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase
B.[see comment]. Science 277, 567-570.
Tan, C., Deardorff, M. A., Saint-Jeannet, J. P., Yang, J., Arzoumanian, A.
and Klein, P. S. (2001). Kermit, a frizzled interacting protein, regulates
frizzled 3 signaling in neural crest development. Development 128, 3665-
Tani, T. T. and Mercurio, A. M. (2001). PDZ interaction sites in integrin alpha
subunits. T14853, TIP/GIPC binds to a type I recognition sequence in alpha
6A/alpha 5 and a novel sequence in alpha 6B. J. Biol. Chem. 276, 36535-
Tohgo, A., Choy, E. W., Gesty-Palmer, D., Pierce, K. L., Laporte, S., Oakley, R.
H., Caron, M. G., Lefkowitz, R. J. and Luttrell, L. M. (2003). The stability of
the G protein-coupled receptor-beta-arrestin interaction determines the
mechanism and functional consequence of ERK activation. J. Biol. Chem. 278,
Wang, L. H., Kalb, R. G. and Strittmatter, S. M. (1999). A PDZ protein regulates
the distribution of the transmembrane semaphorin, M-SemF. J. Biol. Chem. 274,
Yang, J., Tan, C., Darken, R. S., Wilson, P. A. and Klein, P. S. (2002). Beta-
catenin/Tcf-regulated transcription prior to the midblastula transition.
Development 129, 5743-5752.
Yung, Y., Dolginov, Y., Yao, Z., Rubinfeld, H., Michael, D., Hanoch, T.,
Roubini, E., Lando, Z., Zharhary, D. and Seger, R. (1997). Detection of ERK
activation by a novel monoclonal antibody. FEBS Lett. 408, 292-296.
Development 133 (18)