shown that a truncated form of rNTS1 with a deletion of 52 amino
acids from the COOH-terminal end did not affect the ability of the
receptor to achieve phospholipids hydrolysis, but receptor
internalization was dramatically reduced. Recently it has been
shown that NTS1 is able to dimerize in detergent solution in a
concentration-dependent manner . Interestingly, an alterna-
tive splice variant of the calcitonin receptor that lacks the seventh
transmembrane helical bundle causes a dominant-negative effect
on the surface expression of the full-length isoform . Therefore,
even if this receptor isoform is unable to exert a functional
response in the cell, the expression of this truncated variant may
affect the surface expression of the full-length isoform.
isoform would encode a truncated protein of 280 aa
which presents TM1–4, E1–2 and I1–2 encoded by exon 1 equal to
the long isoform (Fig. 3). However,41 aminoacidsencoded by exon3
and the initial 35 nucleotides of exon 4 are speciﬁc to this variant. A
similar isoform has been detected in rat and mouse NTS2, which
share about 60% homology with NTS1. In both, the alternative splice
variant (vNTS2) lacks an internal 181 bp sequence. This deletion
leads to a shift of the reading frame which changes the C-terminal
amino acid sequence leading to a truncated protein of 282 and 281
amino acids, respectively [1,12,19]. Hydrophobicity analysis indi-
cated that rat vNTS2 is a 5-transmembrane domain receptor with an
intracellular C-terminal tail . COS-7 cells transfected with rat
vNTS2 demonstrate that this variant (1) binds to NT although with
considerably lower afﬁnity, (2) presents functional coupling to the
MAPK signaling pathway, (3) associates both with itself and with the
full-length 7-TM receptor to form homo- and heterodimer species
. The sequence identity between vNTS2 and NTS1
60% so it is possible that this truncated isoform may exert some of
the effects observed for rat vNTS2.
would encode a truncated protein of 363 aa and
conserves TM1–4, I1–2 and E1 compared to the full-length isoform
(Fig. 3). However, the E2 loop presents additional amino acids
which are encoded by exon 3 and the 37 nucleotides of intron 3.
Interestingly, the recovery of the open reading frame after intron
inclusion leads to a TM5 equivalent to TM7 of the wild-type form
and an identical carboxyl terminus to that of NTS1 (Fig. 3).
Hydrophobicity analysis using different transmembrane topology
prediction methods available through the Expasy Proteomics
Server  did not identify new TM domains. Although we do not
yet know if this isoform can insert in the plasma membrane and
bind to neurotensin, the C-terminal end is identical to native
protein, and hence it may exert all the functions attributable to this
domain, such as coupling to G
and receptor internaliza-
tion. In fact, deletion of most of the I3 residues of rNTS1 and
expression of the deleted receptor in CHO cells resulted in a total
loss of PLC activation by NT without affecting the ability of the
peptide to stimulate cAMP production .
Finally, the 241 aa protein resulting from the splicing of exon 1
to exon 4 is devoid of the last three transmembrane domains and
presents an extracellular C-terminal end that corresponds to the E2
loop of the long form plus two new amino acids (Fig. 3). The
absence of important structural and functional domains in this
truncated receptor suggests that its ability to insert in the plasma
membrane, bind to NT and activate G protein-coupled pathways
may be impaired.
Androgen deprivation of LNCaP cells in culture gives rise to
synthesis and secretion of neurotensin, in turn inducing increased
cell proliferation and invasion . The presence of truncated
receptor isoforms in PC3 and DU145, two cell lines representing an
advanced stage of the disease, points to new NT signaling in
prostate cancer. Although the NTS1 isoforms described here have
been detected in prostate cancer cell lines, we are already aware of
the presence of these new mRNA isoforms in primary tumors of
myometrium, but not in matched normal myometrium (unpub-
The increasing evidence of peptide receptor overexpression as
well as tumor speciﬁc peptide receptor has stimulated the
development of speciﬁc target, since small radiolabeled peptides
can bind to these receptors and deliver radioactivity to the tumor
cells for cancer diagnosis and/or targeted radiotherapy .
Alternatively, the presence of cancer-related alternative splicing of
transmembrane proteins offers great potential for the selective
therapeutic targeting of tumors .
In conclusion, in this study we describe for the ﬁrst time in the
prostate cancer cell lines PC3 and DU145, four new alternative splice
variants of NTS1 receptor. If these mRNA are ﬁnally translated in
theircorresponding protein,these cancercell lines would co-express
native as well as truncated forms of NTS1 receptor.
The authors are very grateful to Dr. Luz Candenas for the
revision of the manuscript and helpful comments and to Guido
Jones for a careful review of the text. This investigation was
supported in part by the Instituto Canario de Investigacio
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T.A. Almeida et al. / Peptides 31 (2010) 242–247