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J Biol Chem, Vol. 275, Issue 13, 9557-9562, March 31, 2000
From the Institut für Zellbiochemie und klinische
Neurobiologie, Universitätsklinikum Hamburg-Eppendorf,
Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany
Interaction between the C terminus of a
G-protein-coupled receptor and intracellular constituents may represent
a crucial step in regulating signal transduction. To identify potential interacting candidates the C terminus of the somatostatin receptor subtype 1 was used as bait in a yeast two hybrid screen of a human brain cDNA library. We identified the human Skb1 sequence (Skb1Hs) as interacting protein, which is homologous to the yeast protein known
Skb1 to down-regulate mitosis in Schizosaccharomyces pombe via binding to the Shk1 protein kinase; the latter is a homolog to the
mammalian p21cdc42/Rac-activated protein kinases.
Interaction required almost the entire C terminus of the somatostatin
receptor subtype 1 including the conserved NPXXY motif of
transmembrane region seven; in the case of the Skb1Hs most of the N
terminus and an S-adenosylmethionine binding domain were
mandatory, whereas the C terminus was not essential. Interaction was
verified by coexpression experiments in human embryonic kidney cells.
As revealed by immunocytochemical analysis Skb1Hs expressed alone
aggregates in large cytosolic clusters. When coexpressed, receptor
subtype 1 and Skb1Hs were colocalized at the cell surface; these cells
showed a strong increase in somatostatin binding compared with cells
expressing the receptor only. This may suggest that Skb1Hs acts like a
chaperone by correctly targeting the receptor to the cell surface.
Signaling through GPCRs1
is mediated by a limited set of intracellular transducing proteins,
i.e. the heterotrimeric G-proteins, which may link ligand
binding at the receptor to a wide variety of intracellular responses.
Traditionally it has been assumed that all physiological effects of a
given receptor can be explained by the transducing properties of either
the G The five receptors for the neuropeptide somatostatin (called SSTR1-5)
couple predominantly to pertussis toxin-sensitive G-proteins of the
Gi/Go type (5). This may lead to the inhibition
of adenylate cyclase activity or, in the appropriate cellular
background, to the inhibition of voltage-gated calcium channels and the
activation of inwardly rectifying potassium channels (6). However, the signaling repertoire of somatostatin is much wider and may include effects such as the activation of protein-tyrosine phosphatases (7),
which are not easily reconciled with Gi-mediated signaling. For example, somatostatin appears either to enhance or to reduce the
Recently we have initiated a search for proteins that may interact with
SSTRs and might thus modify signal transduction properties of
somatostatin receptor subtypes such as SSTR1 or SSTR2. As a first
result, we have shown that the C terminus of the rat SSTR2 associates
with the PSD-95/discs large/ZO-1 domain of the cortactin-binding protein 1 and that binding of somatostatin to SSTR2 stimulates the
interaction between the receptor and cortactin-binding protein 1 (9).
In addition we have shown that SSTR2 associates also with another
multidomain protein, SSTRIP, which besides a PSD-95/discs large/ZO-1
domain contains several ankyrin repeats, an Src homology 3 domain,
several proline rich regions, and a sterile alpha motif (10).
In extending our studies we report here the results of a yeast two
hybrid screen using the intracellular C terminus of the SSTR1 as the
bait. As the C-terminal sequences of the SSTR are largely divergent, we
expected that proteins interacting with this region may determine the
specificity between the receptor subtypes and effectors such as ion
channels, protein kinases, or phosphatases. Here we show that the C
terminus of the SSTR1 specifically interacts with the human Skb1Hs
protein, which is homologous to the yeast protein Skb1 known to
down-regulate mitosis in Schizosaccharomyces pombe via
binding to the Shk1 protein kinase; the latter is a homolog to the
mammalian p21cdc42/Rac-activated kinases involved
e.g. in cell viability, normal morphology, and mitosis (11,
12). Delineation of the sequence motifs essential for the Skb1Hs and
SSTR1 interaction are provided along morphological and biochemical
evidence that the two proteins interact with each other. When
cotransfected in human embryonic kidney (HEK) cells SSTR1 and Skb1Hs
are colocalized at the cell surface; these cells also exhibited a
greatly increased somatostatin binding compared with cells transfected
with SSTR1 only, suggesting that Skb1Hs may function like a chaperone
or RAMP protein.
Yeast Two Hybrid Screen--
A cDNA fragment (nucleotide
residues 946-1173) coding for the last 77 amino acids of the C
terminus of the rat SSTR1 (13) was cloned into the Gal4-binding domain
containing yeast bait vector pAS-2 (CLONTECH
Laboratories, Inc., Palo Alto, CA). After transformation into the yeast
reporter strain Saccharomyces cerevisiae CG1945
(CLONTECH) a human brain cDNA library in the
Gal4 activation domain fish vector pACTII
(CLONTECH) was screened for interacting proteins
using protocols available from CLONTECH and from
the Gietz laboratory. Of a total of 1.1 × 106
transformants seven true positive clones were identified by the The 3-Amino-1,2,4-Triazol (3-AT) Growth Inhibition Assay--
To
quantify the strength of interaction between the two interacting
constructs the SSTR1 cDNA fragment encoding the C terminus and the
indicated Skb1Hs constructs were coexpressed in yeast cells; the
interaction was measured by analyzing the effect of increasing
concentrations of 3-AT, an inhibitor of the histidine biosynthesis, on
the growth of the cotransformed yeast cells in liquid cultures (15).
Three to six cotransformed yeast colonies grown on
Leu Expression in HEK Cells and Coimmunoprecipitation--
An
expression vector containing an N-terminal T7-epitope tag in frame with
the human hSSTR1-coding sequence (referred to as NT7-tagged SSTR1) was
obtained by cloning the hSSTR1 cDNA into a modified pcDNA3
plasmid (Invitrogen, Leek, The Netherlands) as described (16, 17).
Full-length human Skb1Hs was constructed from the initial clone
(encoding amino acids 55-637) obtained from the yeast two hybrid
library, and an expressed sequence tag clone containing the N terminus
(amino acids 1-54). The cDNA was cloned into modified pcDNA3
vectors coding for either N-terminal or C-terminal c-Myc tags
(Invitrogen). The cDNAs for NT7 tag SSTR1 and the Skb1Hs were then
coexpressed in HEK cells by transient transfection using the calcium
phosphate method (18). For immunoprecipitations, cells were lysed in
radioimmune precipitation buffer (1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM
Tris-HCl, pH 8.0, 10 mM sodium pyrophosphate, 0.2 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 µg/ml pepstatin, 5 mM EDTA) on ice for 10 min and
centrifuged at 15,000 × g for 20 min at 4 °C. The
epitope-tagged receptor was precipitated from the supernatant fraction
using the monoclonal T7 antibody (Novagen, Madison, WI) and protein A-Sepharose (Amersham Pharmacia Biotech) as described (17). Precipitates were analyzed by Western blotting using either T7 or c-Myc
(Sigma) monoclonal antibodies as primary and a goat anti-mouse IgG-coupled to alkaline phosphatase (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA) as a secondary antibody.
Site-directed Mutagenesis--
Deletion mutants of SSTR1 in the
expression vector NT7 tag pcDNA3 were obtained by polymerase chain
reaction using mutagenic primers introducing a stop codon and the
appropriate restriction sites (16). Deletion mutants in the yeast two
hybrid vectors pAS-2 (for SSTR1 mutants) and pACTII (for Skb1Hs
mutants) were obtained similarly by polymerase chain reaction.
Confocal Microscopy--
For the colocalization of human SSTR1
and c-Myc-tagged Skb1Hs (tag at the C terminus), transfected HEK cells
were plated onto poly-D-lysine-coated glass coverslips.
After incubation in normal growth media for 2 days, cells were fixed
and permeabilized as described (16). After incubation with a mixture of
T7 mouse monoclonal antibody (1:10,000) and SSTR subtype-specific
rabbit polyclonal antisera (1:2000) in phosphate-buffered saline
containing 2% normal fetal calf serum overnight at 4 °C,
cy2-conjugated goat anti-mouse and cy3-conjugated goat anti-rabbit were
used as secondary antibodies (9). Cells were viewed with a confocal
microscope (Leitz laser scanning microscope) using specific settings
for cy2 (argon laser/excitation at 488 nm/emission band pass 510-525 nm) and cy3 (helium-neon laser/excitation at 543 nm/emission low pass
570 nm). The absence of bleed through between the two fluorophores was
verified by performing parallel staining experiments on cells expressing either one or the other antigen only.
Somatostatin Binding Assay--
Binding assays were performed on
intact cells, transiently transfected with various cDNAs, plated on
poly-D-lysine-coated 24-well plates (100,000 cells/well)
one day before the experiment. For the binding experiment, the cells
were washed once with ice-cold phosphate-buffered saline and incubated
with 300 pM 125I-SST14 (2200 Ci/mmol, Amersham
Pharmacia Biotech) in serum-free growth medium on ice for 2 h. The
binding reaction was stopped by washing four times with ice-cold
phosphate-buffered saline. Cells were lysed by treatment with 0.2 M NaOH/1% sodium dodecyl sulfate for 10 min at room
temperature and transferred into a Identification of an SSTR1-interacting Protein--
To identify
potential SSTR1-interacting proteins the C terminus of SSTR1 (starting
with the amino acid sequence SCANPILY in the C-terminal part of
transmembrane region 7 and thus including the canonical
NPXXY motif) was used as a bait in a yeast two hybrid screen. Seven positive clones were obtained from a human brain cDNA
library four of which showed only a weak response in the Delineation of the Interacting Regions of Skb1Hs and
SSTR1--
The structure of the Skb1Hs protein exhibits only one
obvious motif, the S-adenosylmethionine binding domain,
which does not allow any conclusions regarding its interaction with the
C terminus of SSTR1 (Fig. 1A).
In an attempt to delineate the regions involved in the interaction of
the two partners, several deletion mutants were generated from the
initial target vector Skb1Hs obtained from the human brain cDNA
library; they were cotransformed with the SSTR1 fragment in yeast cells
which grow on His-deficient plates only when cells contained both
interacting partners. Growth in the presence of 30 mM 3-AT,
an inhibitor of the histidine biosynthesis pathway, reflects a
significant interaction between the two interacting constructs. Fig.
1B shows that interactions were strongest with the initial
Skb1Hs construct (amino acid residues 55 to 637) and with a full-length
Skb1Hs clone; the N-terminal region up to amino acid residue 55 was not
essential for interaction. Deletions were tolerated at the C terminus
up to the AdoMet binding domain (amino acid residues 55-480). Although
growth of cotransformed yeast cells containing a Skb1Hs construct with
a modified AdoMet binding domain (amino acid residues 55-438) was
markedly reduced, the AdoMet binding domain itself (amino acid residues
337-480) was not sufficient to trigger growth. Deletions toward the
N-terminal part of the initial Skb1Hs construct (e.g. amino
acid residues 163-637) also abolished the interaction with SSTR1 as
evidenced by the lack of growth on 3-AT plates. Positive interactions
between the interacting partners were verified by the
To delineate the C-terminal receptor interacting region, yeast cells
cotransformed with Skb1Hs and various SSTR1 constructs were analyzed by
the 3-AT growth inhibition assay (Fig.
2A). Cells were grown in
His-deficient medium in the presence of various 3-AT concentrations,
and the strength of the interactions quantified as the relative
resistance to 3-AT was expressed as IC50 values (Fig. 2,
B and C). Only the initial
SSTR1316-391 construct showed strong growth response with
an IC50 of 33.5 mM 3-AT (Fig. 2, B
and C), whereas deletions at the N terminus
(SSTR1322-391 and SSTR1328-391) resulted in
an increase in the sensitivity to 3-AT with an IC50 of 5.6 and below 2 mM 3-AT, respectively. The twelve amino acid
residues of the N terminus of the initial SSTR1316-391
construct are apparently essential for the full interaction with the
Skb1Hs protein. This region includes the highly conserved NPILY
motif in the seventh transmembrane region also present in other SSTR
subtypes such as SSTR2; yet the C-terminal SSTR2305-369
construct displayed no measurable interaction with the Skb1Hs protein
indicating that this region is not exclusively sufficient for
interaction. Indeed, a deletion at the C terminus of SSTR1
(SSTR1316-352) significantly decreased the interaction
with the Skb1Hs protein. Thus our data demonstrate that the interaction
with Skb1Hs requires N- and C-terminal regions of the
SSTR1316-391 construct including the conserved NPILY
motif.
Interaction of the Human SSTR1 with the Skb1Hs Protein in HEK
Cells--
To investigate the interaction between human SSTR1 and
Skb1Hs in a mammalian system the full-length constructs were
cotransfected in HEK cells. For identification the proteins carried
either a T7 tag at the N terminus of SSTR1 or a c-Myc tag at the C
terminus of Skb1Hs. When the receptor was immunoprecipitated from
lysates of cotransfected HEK cells using a T7 monoclonal antibody,
analysis of the resulting immunoprecipitate by Western blotting
revealed the presence of c-Myc-tagged Skb1Hs as identified by a band at 72 kDa. This band was absent when the SSTR1 was omitted from the transfection mixture (Fig.
3A). Similar results were
obtained with a Skb1Hs construct carrying the c-Myc epitope tag at the N terminus (data not shown). As already inferred from the strong
As indicated already by experiments in the yeast system, no interaction
between SSTR2 and Skb1Hs was observed in coprecipitation experiments
(Fig. 3B). Similarly, coexpression of a NT7 epitope-tagged µ-opioid receptor did not lead to an interaction between this receptor and the Skb1Hs protein. However, NT7-tagged SSTR4, which is
most similar to the C terminus of SSTR1 in its primary structure (Table
I), was able to interact with
c-Myc-tagged Skb1Hs in HEK cells (Fig. 3B).
To verify the specificity of the interaction between the C-terminal
regions of the SSTR1 and Skb1Hs, several deletion mutants of the
NT7-tagged SSTR1 C terminus were constructed and used for coexpression
in HEK cells. In coimmunoprecipitation assays the construct NT7-tagged
SSTR11-370 lacking the last 21 amino acid residues
efficiently precipitated the c-Myc-tagged Skb1Hs protein, whereas
further truncations at the C terminus (NT7-tagged SSTR11-343 or NT7-tagged SSTR11-327)
abrogated the interaction with Skb1Hs indicated by the absence of the
latter in the immunoprecipitate (Fig.
4A). These results are
consistent with those obtained in the yeast two hybrid system (Fig. 2).
Similar results were obtained when c-Myc-tagged Skb1Hs was
precipitated, and T7-tagged SSTR1 deletion mutants were detected by
Western blotting using the T7 antibody. Only full-length NT7-tagged
SSTR11-391 and NT7-tagged SSTR11-370 were
observed in the precipitates, whereas shorter deletion constructs were
not detected (Fig. 4B).
Confocal microscopic analysis of HEK cells transfected with
c-Myc-tagged Skb1Hs alone revealed that the protein was found exclusively in intracellular clusters, which do not resemble any known
cellular structures (Fig. 5). Such
intracellular clusters or aggregates have been observed in expression
experiments in HEK cells, for instance when cells were transfected with
proteins involved in the intracellular anchoring of ligand-gated ion
channels (22). This clustering may reflect an atypical aggregation of proteins because of their high expression level and/or to the absence
of appropriate interacting proteins. In the case of Skb1Hs a dramatic
change in the fluorescence pattern was observed when the latter was
coexpressed with NT7-tagged SSTR1. Whereas some of the immunostaining
for Skb1Hs still remained in those clusters, fluorescence signals could
also be observed in a diffuse distribution throughout the cytosol as
well as concentrated at the plasma membrane where it colocalizes with
SSTR1-specific fluorescence. None of these changes were observed when
the Skb1Hs protein was cotransfected with the SSTR2 (Fig. 5).
To investigate the function of the interaction of Skb1Hs with the C
terminus of SSTR1, we examined the number of binding sites for
radioactively labeled somatostatin when SSTR1 was expressed in HEK
cells in the absence or presence of Skb1Hs. Binding assays demonstrated
a dramatic increase of specifically bound radioligand to those cells
cotransfected with SSTR1 and Skb1Hs compared with cells transfected
with SSTR1 only (Table II). These results
suggest that the interaction between Skb1Hs and SSTR1 is an important step for transporting the receptor to the cell surface.
The data presented here clearly identify a specific interaction
between the C terminus of SSTR1 and the human homolog of a kinase-binding protein from the fission yeast S. pombe. When
trying to identify the regions of both proteins involved in this
interaction, we determined that large portions of the bait as well as
of the fish were necessary to obtain a strong response in the yeast two hybrid system. This suggests that the interaction is not mediated by a
single domain but that the intact tertiary structure of the whole
Skb1Hs protein may be required to bind the SSTR1 C terminus.
In SSTR1, only the extreme C terminus could be deleted without
abolishing the interaction between both proteins. Regions necessary for
the interaction are located close to or even within the seventh transmembrane region including the NPXXY motif common to
many GPCRs. It is interesting to note that SSTR4 was the only other receptor tested that could be coimmunoprecipitated with the Skb1Hs protein. In respect to its C terminus, SSTR4 is the subtype with closest homology to the SSTR1; the homology extends up to the amino
acid position 360 in the SSTR1 sequence (Table I), a region essential
for the interaction of SSTR1 with Skb1Hs both in yeast and in HEK cells.
The function of the Skb1 protein is unclear at present in the yeast as
well in human cells. In S. pombe, it was suggested that
Skb1, through its interaction with the Shk1 kinase, might influence
mitogen-activated protein kinase pathways and the organization of the
cytoskeleton, leading to changes in cell morphology (11). When we
analyzed activation of the mitogen-activated protein kinase by
somatostatin in HEK cells expressing SSTR1, we observed efficient activation of this pathway regardless if Skb1Hs was coexpressed or not
(data not shown). However, HEK cells express endogenous Skb1Hs
(21), which might be sufficient to fulfill a role in SSTR1 signal transduction.
Skb1Hs, as shown here in cells overexpressing this gene, is localized
in undefined cytosolic structures. This localization may be a
consequence of the lack of appropriate interaction partners within the
transformed cells, as has been observed before for proteins involved in
the targeting of other neurotransmitter receptors (22). Accordingly, a
dramatic relocalization of Skb1Hs was observed in cells coexpressing
SSTR1, leading to the detection of Skb1Hs-specific fluorescence at the
plasma membrane where it colocalizes with SSTR1.
The function of the novel interaction reported here remains unclear at
present; with respect to signal transduction by SSTR1, Skb1Hs has not
been able to confer the ability to couple to a G-protein gated inwardly
rectifying potassium channel when coexpressed in Xenopus
oocytes. However, our findings may present an explanation of several
inconsistencies in the literature. Thus functional coupling of SSTR1 to
the adenylate cyclase system has been observed in some but not all
experiments (23-25). In addition, inhibition of voltage-gated calcium
channels by SSTR1 has been disputed (26, 27). These discrepancies may
be attributed to the cellular environment, in particular to the
presence of the Skb1Hs protein. Our data show that coexpression of
Skb1Hs augments functional expression of the SSTR1 protein at the cell
surface. Presumably Skb1Hs may be required for efficient targeting of
the receptor to the cell surface; low expression levels of Skb1Hs may
interfere with the functional insertion of SSTR1 into the plasma
membrane supporting the idea that Skb1Hs functions as a chaperone- or
RAMP-like protein. Interestingly, Skb1Hs has been found to be tightly
associated with the protein pICln (21); expression of pICln in
Xenopus oocytes regulates the appearance of a swell-induced
chloride channel in the oocyte membrane (28, 29). Thus pICln and Skb1Hs
may play a more general role in the expression of membrane proteins.
We thank Dr. Stefan Schulz, University of
Magdeburg, for rabbit polyclonal antisera against SSTR subtypes.
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant SFB 545/B7 (to H.-J. K. and D. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Inst. für
Zellbiochemie und Klinische, Neurobiologie, Universität Hamburg,
Martinistrasse 52, 20246 Hamburg, Germany. Tel.: 49-40-42803-3344; Fax:
49-40-42803-4541; E-mail: richter@uke.uni-hamburg.de.
The abbreviations used are:
GPCR, G-protein-coupled receptor;
RAMP, receptor activity-modifying protein;
SSTR, somatostatin receptor;
Skb1Hs, Skb1 human sequence;
AdoMet, S-adenosylmethionine;
HEK cells, human embryonic kidney
cells;
3-AT, 3-amino-1,2,4-triazol;
NT7, N-terminal T7.
Interaction of the Somatostatin Receptor Subtype 1 with the Human
Homolog of the Shk1 Kinase-binding Protein from Yeast*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- or G
-subunits that are activated by the receptor.
However, recently evidence has accumulated that intracellular factors
other than the G-protein subunits can affect the outcome of
GPCR-mediated signaling. Thus the 2-adrenergic receptor
associates in an agonist-dependent manner with the
PSD-95/discs large/ZO-1 domain of the Na+/H+
exchanger regulatory factor leading to an activation of exchange activity instead of the inhibitory effect that would usually be expected from a receptor coupled to stimulatory G-proteins (1). Similarly, the interaction of metabotropic glutamate receptors with the
homer protein has been suggested to have an influence on signal
transduction (2). In separate studies, it was shown that accessory
proteins, receptor activity-modifying proteins (RAMPs), regulate the
transport and the pharmacological properties of certain GPCRs such as
the receptor for the calcitonin gene-related peptide; in the presence
of RAMP1 the receptor functions as a calcitonin gene-related peptide
receptor, with RAMP2 as an adrenomedullin receptor (3, 4).
-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid/kainate receptor-mediated response to glutamate in vivo depending on
whether the cells express SSTR1 or SSTR2, respectively (8).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase filter lift assay (14). The specific interaction of
the positive clones with the C terminus of SSTR1 was verified by a
negative control using a cDNA fragment coding for the C terminus of
the rat SSTR2. DNA isolated from the positive clones was sequenced by
the dideoxy chain termination method in an automatic DNA sequencer (Applied Biosystems 377, Weiterstadt, Germany).
/Trp/His plates were
transferred to His-deficient media without 3-AT and grown to 1-1.2
A600 at 30 °C. Only cells containing both
interacting partners grow in His-deficient medium. Identical amounts of
cotransformed yeast cells were inoculated in His media
containing various concentrations of 3-AT. Controls were run with an
SSTR2 construct. After three days at 30 °C the cultures were
measured at 600 nm; the ratios of the ODs obtained in the presence or
absence of 3-AT were calculated, and the IC50 values were
determined. The values reflect the strength of the interaction between
the two interacting constructs; these increased as more 3-AT was
needed to inhibit growth.
-counter for the determination of
cell-associated radioactivity. Nonspecific binding was determined in
the presence of 1 µM unlabeled SST14.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase filter assay; sequencing revealed that they were either identical with syntrophin 2 (19) or similar to a protein interacting with the viral oncoprotein Tax (20) or exhibited sequences
not listed in the GenBankTM data base (two clones). The
remaining three clones with identical inserts were identified as the
human Skb1Hs protein (12, 21), which is structurally and functionally
homologous to the yeast protein Skb1; the latter has been shown to
negatively regulate the mitosis in the fission yeast S. pombe (11, 12). The Skb1Hs clone contained almost the full-length
coding sequence of 637 amino acids lacking only 55 residues at its N
terminus. The interaction of SSTR1 with Skb1Hs was by far the
strongest; in -galactosidase filter assays a significant response
could usually be obtained within a few minutes, whereas for the other
clones a reaction time of more than an hour was necessary to obtain a
signal of similar intensity (data not shown).
-galactosidase assays confirming that most of the Skb1Hs protein is involved in the
interaction with the C terminus of SSTR1.
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Fig. 1.
Analysis of the interaction of Skb1Hs
truncation constructs with human SSTR1. Interactions were analyzed
by transformation of yeast cells with pAS2-hSSTR1 and deletion
constructs of Skb1Hs in pACTII as indicated. A, the
positions of the first and last amino acids of each construct are
shown. M, methionine; L, leucine.
SAM-bd, S-adenosylmethionine binding domain.
B, growth on Leu/Trp/His-deficient 3-AT containing plates is
indicated by +++, ++, or + depending on the size and the number of the
colonies obtained; -, no growth; n.d., not determined. The presence of
an equal number of transformants was controlled for each transformation
by plating an aliquot on Leu/Trp-deficient plates. The results were
confirmed by a semiquantitative -galactosidase assay.
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Fig. 2.
Delineation of the region in the SSTR1 C
terminus required for interaction with Skb1Hs. A,
interactions were analyzed by transformation of yeast cells with
pACTII- Skb1Hs and pAS2-SSTR1 deletion mutants as indicated. Numbers of
the first and last amino acids of the constructs are shown. As a
negative control the corresponding region of SSTR2 in the pAS2-vector
was used. TM, transmembrane region. B, growth
inhibition assay in liquid culture. Shown are the growth inhibition
curves of SSTR1 deletion constructs with progressive truncation at the
N terminus. The dose-response data were subjected to nonlinear
regression analysis using the GraphPad Prism software (GraphPad Inc.,
San Diego, CA). Yeast transformants expressing both plasmids were
cultured in His-deficient liquid medium containing various
concentrations of 3-AT. The optical density of the culture at 600 nm
after 3 days is given as the fraction of growth obtained in the absence
of 3-AT. Values are the mean ± S.D. of triplicate determinations.
C, list of IC50 values calculated from
B. No exact IC50 values could be calculated for
the SSTR2305-369 and SSTR1328-391 constructs
because of the low affinity of the interaction.
-galactosidase reaction in the yeast two hybrid experiment, the interaction between SSTR1 and Skb1Hs appeared to be very strong because
it could still be observed when lysis of the cells was carried out in
the presence of 1% SDS.
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Fig. 3.
Coimmunoprecipitation of human SSTR1 and
Skb1Hs. A, HEK cells were transfected either with
Skb1Hs cDNA alone or in combination with NT7-tagged SSTR1 cDNA.
After lysis in radioimmune precipitation buffer containing either 0.1%
or 1% SDS, the receptor was precipitated using the T7 antibody;
precipitates were then analyzed by Western blotting using either the
c-Myc antibody (left panel) or the T7 antibody (right
panel). B, HEK cells were transfected with Skb1Hs
cDNA alone or in combination with cDNAs encoding either
NT7-tagged SSTR1, -2, -4, or the rat µ-opioid receptor. After lysis
and immunoprecipitation as described in A, Western blotting
was performed with c-Myc (upper panel) and T7 (lower
panel) antibodies. IP, immunoprecipitation;
IB, immunoblotting; marker, full range rainbow
protein molecular weight marker (Amersham Pharmacia Biotech);
open arrows indicate the position of the precipitated Skb1Hs
protein at 72 kDa; SSTR1 appears in multiple bands; the lowest band (68 kDa) presumably corresponds to a monomeric receptor (calculated
molecular mass, 43 kDa), which is glycosylated. In addition,
filled arrow heads indicate positions of 165-, 155-, and
105-kDa proteins representing most likely differentially glycosylated
receptors (or multimers) (A). SSTR2, the rat
µ-opioid receptor, and SSTR4 also appear as several differentially
glycosylated proteins migrating as distinct bands (B).
Amino acid sequence comparison of the C terminus of SSTR1 and -4
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Fig. 4.
Coimmunoprecipitation of Skb1Hs with human
SSTR1 deletion constructs. A, immunoprecipitation
experiments from HEK cells coexpressing Skb1Hs and various SSTR1
constructs were performed as described before. Upper and
middle panels, detection of the c-Myc-tagged Skb1Hs protein
in cellular lysates and precipitates using the c-Myc antibody.
Lower panel, detection of the various NT7-tagged SSTR1
proteins in immunoprecipitates. SSTR1 immunoreactivities are visible in
several diffuse bands ranging from molecular mass of 65-160 kDa;
truncated receptor proteins of 55 (*) and of 52 kDa (**) are observed.
B, precipitation was performed using the c-Myc antibody, and
immunoblots were probed with c-Myc (upper panel) and T7
(lower panel) antibodies. IP,
immunoprecipitation; IB, immunoblotting.
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Fig. 5.
Colocalization of human SSTR1 and Skb1Hs in
HEK cells. Cells expressing either (i) c-Myc-tagged Skb1Hs, (ii)
c-Myc-tagged Skb1Hs and SSTR1, (iii) c-Myc-tagged Skb1Hs and SSTR2, or
only (iv) SSTR1 were stained with mouse anti-c-Myc and the appropriate
rabbit anti-SSTR antibodies. Cells were examined by confocal
microscopy, and optical sections were obtained from the center of the
cells. In the upper panel SSTR1-specific fluorescence
signals were recorded from the cy2 (anti-rabbit), and in the
lower panel Skb1Hs signals from the cy3 specific channel
(anti-mouse) are shown. Colocalization of SSTR1-specific
(green) and Skb1Hs-specific (red) signals is
indicated by the occurrence of yellow staining in the
lowest panel. Note the absence of staining with the
respective antibodies when either Skb1Hs or the SSTR1 was omitted from
the transfection mixture; in these cases, the contours of the cells are
indicated. Bar, 5 µm.
Specific binding of 125I-SST14 to transfected HEK cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
The data presented here form part of a thesis by A. S.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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