(Received for publication, June 2, 1997, and in revised form, June 24, 1997)
From the Many proteins involved in
SV1 exocytosis have been
recently identified, but still little is known about the signals that
control this highly regulated process (1). The main release stimulus is
Ca2+, which enters nerve terminals through
voltage-sensitive Ca2+ channels. However, neuroexocytosis
can be stimulated in the absence of extracellular Ca2+
too.
One of such Ca2+-independent activators of neurosecretion
is LTX, a Black Widow spider presynaptic neurotoxin that causes massive exocytosis of SV (reviewed in Ref. 2). The toxin mode of action remains
controversial. On the one hand, LTX can make cation-permeable channels
in lipid bilayers (3) and can dramatically increase ion fluxes through
the plasma membrane of toxin-sensitive cells (4), thus triggering
neurotransmitter release. In most systems, the influx of
Ca2+ indeed plays an important role in LTX effect (2).
Whether the toxin itself forms pores in the cell membrane or induces
the opening of some endogenous channels is still unknown. On the other hand, the toxin has been shown to act in the absence of extracellular Ca2+ (5-7) and other divalent cations (8). In addition, to
be able to act, LTX requires specific membrane receptors, which are found in the plasma membrane of neurons and some endocrine cells (2).
In neuromuscular junctions the toxin receptors are localized presynaptically (9). The existence of two types of receptors, Ca2+-dependent and -independent, has long been
recognized (6), the latter ones being solely responsible for the toxin
action in the absence of Ca2+. To elucidate the LTX mode of
action and better understand the mechanism of neurosecretion, it is
important to know the structure of the LTX receptor.
In an attempt to isolate the receptor, neurexins, neuronal cell surface
proteins, were discovered (10). However, neurexins interact with the
toxin only in the presence of Ca2+ (11) and thus may not be
responsible for LTX effect in the absence of this ion. Moreover, LTX
potently causes dopamine release in a PC12 cell line that completely
lacks Ca2+-dependent receptors (6), thus ruling
out the importance of neurexins in the toxin action. Recently, we
purified latrophilin, a synaptic protein that binds LTX independently
of Ca2+ (12). The isolation of the same protein was later
also described by others (13). Here we report the cloning and
sequencing of latrophilin, the Ca2+-independent LTX
receptor, and demonstrate that it is a novel member of the
secretin/calcitonin family of G protein-coupled receptors. Our findings
implicate G proteins in regulation of neurotransmitter release.
High purity LTX was
purchased from Neurogen, UK. Latrophilin from rat brain was isolated by
LTX affinity chromatography (12) and further purified by SDS-PAGE. The
protein was digested in gel pieces with endoproteinases Lys-C or
trypsin (Wako). Peptides were recovered by sonication and applied
directly to an Aquapore AX-300 column (2 × 0.5 mm, custom made)
and a Reliasil C18 column (150 × 1 mm) in series on a
Michrom Ultra Fast Protein Analyser HPLC system. The columns were
developed with a linear acetonitrile gradient in 0.1% trifluoroacetic
acid at a flow rate of 50 µl/min. Collected fractions were applied to
a high sensitivity Procise system (Applied Biosystems) employing a
capillary HPLC C18 column (250 × 0.8 mm). Initial
yields were in the range of 0.5 to 5 pmol.
A
degenerate oligonucleotide probe,
AA(A/G)TACGACCT(C/G)CG(C/G)ACCCG(C/G)AT(C/T) AA-3 The insert from clone
RBCR9-15 was recloned into a mammalian vector pcDNA3 (Invitrogen).
COS-7 cells (European Cell Culture Collection) were transiently
transfected with this construct or the vector alone, using SuperFect
reagent (Qiagen) and 10 of µg DNA/10-cm plate. Cells were harvested
after 1-4 days and washed with phosphate-buffered saline containing 20 mM EGTA, and aliquots (~106 cells) were
incubated with different concentrations of iodinated LTX for 15 min. A
100-fold excess of unlabeled LTX was included in controls. Unbound
toxin was removed by filtration. For immunostaining, ~5 × 106 transfected cells were solubilized in 2% Thesit and
subjected to WGA affinity chromatography as described (12) followed by SDS-PAGE and Western blotting.
Northern blots (Bios Laboratories) of total
RNA from rat tissues were hybridized to a randomly radiolabeled insert
from clone RBCR9-15. Latrophilin preparations enriched in
heterotrimeric G proteins were made as published (12), except that 20 µM Mg2+ and 2 mM GDP were
included during purification. Antibodies against G protein To determine the structure of latrophilin, we purified
approximately 300 µg of rat brain latrophilin. Fourteen peptides were isolated from the protease-digested protein. The peptide sequences were
used to design degenerate oligonucleotide probes for the screening of a
rat brain cDNA library. As a result of this screening, we isolated
20 cDNA clones, all of which overlapped and represented a piece of
cDNA ~8.3 kb long. The longest clone (RBCR9-15; 5.7 kb)
contained a 4.4-kb open reading frame and encoded all peptides obtained
from rat brain latrophilin. The deduced primary structure of this
protein, termed rat latrophilin 1 (LPH1), is shown in Fig.
1A.
The LPH1 molecule (Fig. 1B) comprises the following putative
domains: an 849-residue-long extracellular domain, seven hydrophobic transmembrane segments (TMSs) and a cytoplasmic tail of 372 amino acids. The extracellular domain begins with a hydrophobic signal peptide. This is followed by a cysteine-rich stretch homologous to
galactose-binding lectin (14), an extended region of homology to
olfactomedin (15, 16) (Fig. 2,
A and B), a proline/threonine-rich domain, and a
glycosylated spacer fragment. The presumably cytoplasmic C-terminal
portion of LPH1 contains five cysteine residues. Three of these are
positioned very similarly to cysteines in the cytoplasmic tail of
rhodopsin and may also be palmitoylated (17). There are several
possible phosphorylation sites in the molecule (Fig. 1A)
that may play a role in receptor desensitization in response to
endogenous ligand(s) under physiological conditions. Proline residues,
abundant in this region, could interact with SH3 domains of proteins
involved in signaling.
The main attribute of LPH1 is the presence of seven TMSs that are
25-45% identical to the corresponding parts of several recently characterized proteins (18-30) (Fig. 2C). These proteins
belong to the secretin/calcitonin family of GPCRs; most bind peptide hormones and are coupled via heterotrimeric G proteins to the stimulation of various release processes. LPH1 possesses many of the
features thought to be important for GPCRs, such as a negatively charged amino acid in TMS III, conserved cysteines in
extracellular loops 1 and 2 that may form a disulfide bond, and proline
residues in TMS IV and TMS V. LPH1 is the longest
known GPCR and, together with its closest homologues (Fig.
2C), belongs to a subfamily of "long seven-TMS
receptors."
In good agreement with our previous results on the tissue distribution
of latrophilin (12), LPH1 mRNA has been found in the brain but not
in non-neuronal tissues (Fig.
3A). We have also previously
shown that latrophilin is greatly enriched in synaptosomal plasma
membranes (9). This and the strictly presynaptic mode of toxin action
suggests that latrophilin is a presynaptic protein. Neuromuscular
junctions, the primary target of LTX, also display abundant toxin
receptors (9). However, muscle cells do not transcribe any latrophilin
message (Fig. 3A), corroborating the mainly presynaptic
localization of this protein. Apart from the nervous system, LTX
receptors are present in chromaffin and PC12 cells (2) and some
pancreatic
Although all peptides from latrophilin have been found in the structure
of LPH1, we carried out experiments directed at proving that LPH1 is
indeed the toxin receptor. Full-length LPH1 was expressed in COS cells
using an appropriate vector. Upon transfection with this construct but
not the vector alone, cells specifically bound iodinated LTX in the
absence of Ca2+. The number of binding sites grew until day
3 (Fig. 3B). Analysis of these binding sites revealed that
LTX and expressed latrophilin interact with high affinity
(Kd = ~2.5 nM) (Fig. 3C). This value is higher than that of LTX receptors in brain (12), possibly
due to a different protein context at the surface of COS cells. The
plasma membranes from transfected cells were then analyzed by WGA
affinity chromatography and immunoblotting (Fig. 3D).
Latrophilin expressed in COS cells was indistinguishable from the brain
protein. These observations unequivocally prove that latrophilin is the
Ca2+-independent LTX receptor and confirm our previous
conclusion that it alone is sufficient for high affinity LTX binding
(12).
Our finding that full-length latrophilin has seven TMSs and provides
binding sites for the toxin suggested that neurotransmitter release may
be controlled through a receptor-mediated activation of heterotrimeric
G proteins. Such a mechanism is well known to regulate exocytosis in
secretory cells, although the precise point of the G protein action in
the docking/fusion process has not been established yet. G proteins are
highly enriched in synapses (31), and the basic mechanism of exocytosis
regulation in neurons should be similar to that in other secretory
cells. Despite this, the idea that heterotrimeric G proteins may be
implicated in SV exocytosis is relatively new (32, 33) and has not
become widely accepted yet (1). Interestingly, LTX has already been
shown to stimulate (in the presence of Ca2+) the production
of IP3 in PC12 cells (34), a function usually controlled by
GPCRs. Accordingly, in LPH1-transfected cells but not in control cells,
10 nM LTX causes up to 2-fold elevation of cAMP and
IP3 (Fig. 4A). Although this effect was observed
only in 10 µM Ca2+, it was not mimicked by a
Ca2+ ionophore A23187, suggesting a functional coupling
between the LTX receptor and G proteins, probably enhanced by
Ca2+ entry into the cells (see also Ref. 34). Therefore, to
further confirm a putative functional link, we demonstrate that
latrophilin physically interacts with a G protein
We also examined what type of second messenger may be involved in the
LTX-evoked norepinephrine secretion in neurons. However, our
experiments on synaptosomes4
indicate that the main second messengers, cAMP, cGMP, and
IP3 do not control SV exocytosis and do not play a major
role in the toxin action in zero Ca2+. These results appear
to be at variance with the effect of LTX in COS cells expressing LPH1
(Fig. 4A). However, functional links of a GPCR in
heterologous systems may often be different and depend on the
repertoire of heterotrimeric G proteins expressed in a given cell (36).
What could then be the mechanism of LTX action in neurons, provided its
effect is mediated by latrophilin, a GPCR? G proteins have recently
been shown to control neurotransmitter release by regulating
preferentially those presynaptic calcium channels that are directly
associated with release sites, indicating that channel components of
the release sites may be effectors for G proteins (33). Although the
toxin opens Ca2+-permeable channels in the plasma membrane
of sensitive cells, these differ both from pores made by LTX in lipid
bilayers (37) and from conventional Ca2+ channels (38, 39).
LTX also induces fluxes of other cations in synaptosomes and
neuroblastoma cells (40, 41). This implicates the involvement of
nonspecific cation channels that have been found in numerous cell types
(42). Such channels are also controlled by G proteins (43) and could
participate in regulation of secretion. On the other hand, in some
systems the toxin action may proceed without ion fluxes and membrane
depolarization (44)2 and may be based on the activation by
G proteins of components of release sites different from ion channels.
It is important to stress, however, that the site of G protein action
in exocytosis is still unknown both in endocrine cells and at nerve
terminals.
Taken together, our findings strongly suggest that SV exocytosis may be
controlled by the presynaptic machinery that links receptors to G
proteins to release sites and bypasses the main second messengers. This
streamlined type of coupling is already known for ligand-evoked
regulation of hormone secretion (45). Consequently, like hormone
release, neurosecretion could be directly controlled by extracellular
ligands. Remarkably, the N-terminal extracellular domain of LPH1 is
homologous to olfactomedin, a protein of olfactory epithelium
implicated in odorant binding (15), and to olfactomedin-related
proteins found throughout the brain (16) (Fig. 2B). These
soluble proteins may bind some ligands and deliver them by diffusion to
GPCRs in the plasma membrane. The striking feature of latrophilin is
that this olfactomedin-like domain is attached to a membrane-bound
GPCR, suggesting an evolutionary adaptation for a more efficient
ligand search and signal transduction. The finding of endogenous
latrophilin ligands should therefore reveal new ways of regulating
neuroexocytosis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U78105. We thank J. O. Dolly and P. Foran for
providing antibodies and helpful discussions, J. J. Hsuan for help
with protein sequencing, Phase Separations for the capillary ion
exchange column, and M. Woodland for DNA sequencing.
Department of Biochemistry,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Latrotoxin (LTX) stimulates massive
exocytosis of synaptic vesicles and may help to elucidate the mechanism
of regulation of neurosecretion. We have recently isolated latrophilin,
the synaptic Ca2+-independent LTX receptor. Now we
demonstrate that latrophilin is a novel member of the secretin family
of G protein-coupled receptors that are involved in secretion. Northern
blot analysis shows that latrophilin message is present only in
neuronal tissue. Upon expression in COS cells, the cloned protein is
indistinguishable from brain latrophilin and binds LTX with high
affinity. Latrophilin physically interacts with a G
o
subunit of heterotrimeric G proteins, because the two proteins
co-purify in a two-step affinity chromatography. Interestingly,
extracellular domain of latrophilin is homologous to olfactomedin, a
soluble neuronal protein thought to participate in odorant binding. Our
findings suggest that latrophilin may bind unidentified endogenous
ligands and transduce signals into nerve terminals, thus implicating G
proteins in the control of synaptic vesicle exocytosis.
Latrophilin Purification and Sequencing
,
corresponding to the sequence of peptide KYDLRTRIK, was used to
screen a rat brain cDNA library in
ZAPII (~2 × 106 recombinants, Stratagene). cDNA inserts were
sequenced using a Dye Terminator Cycle Sequencing chemistry and an
automated DNA sequencer, Prism 377 (Applied Biosystems). On-line data
base searches were performed using FASTA and BLAST programs
(Intelligenetics). Sequences were analyzed with the Lasergene software
(DNA Star).
-subunits
and synaptotagmin were from Santa Cruz and Affiniti. Antibodies against
SNAP-25, syntaxin, and synaptobrevin were kindly provided by Drs.
J. O. Dolly and P. Foran. Proteins were analyzed by SDS-PAGE,
transferred onto Immobilon membrane (Millipore), and visualized using
respective antibodies and Chemiluminescence Substrate System
(Pierce).
Fig. 1.
The structure of rat latrophilin 1. A, the deduced amino acid sequence of rat LPH1 (GenBankTM
accession number U78105). B, domain model of LPH1. The
signal peptide (SP in B) is shown in
bold. Regions of homology to galactose-binding lectin and
olfactomedin are dotted and shaded, respectively.
Peptide sequences from rat latrophilin are doubly
underlined. The transmembrane domains are presented as black
bars. Branched structures indicate hypothetical N-glycosylation sites. Putative phosphorylation sites for
cAMP-dependent protein kinase (Thr-1155 and Ser-1439),
protein kinase C (Ser-1028, Ser-1034, Ser-1291, and Ser-1343), and
casein kinase II (Ser-1310) are boxed. Underlined
are proline-rich regions (horizontally hatched and marked
Pro in B). Cysteines are shown as bars
in B. Two cysteines, presumably engaged in a disulfide bond,
are marked with asterisks. Zigzag lines denote
hypothetical cysteine isoprenylation (Cys-1231 and Cys-1356) or
palmitoylation (Cys-1105 and Cys-1111 or Cys-1112).
[View Larger Version of this Image (51K GIF file)]
Fig. 2.
Homologies of latrophilin to known proteins.
A, alignment of rat LPH1 with galactose-binding lectin from
sea urchin (14) and a putative protein encoded by Caenorhabditis
elegans cosmid B0457 (GenBankTM accession number Z54306).
B, alignment of LPH1 with bullfrog olfactomedin, OLFM (15),
and rat olfactomedin-related protein, OLFR (16). C,
alignment of the TMS region of latrophilin 1 with corresponding parts
of the secretin family GPCRs: leukocyte activation antigen, CD97 (18);
epidermal growth factor module-containing, mucine-like receptor
(EMR1) (19); vasoactive intestinal peptide receptor
(VIPR) (20); pituitary adenylate cyclase-activating polypeptide receptor (PACR) (21); secretin receptor
(SCRC) (22); glucagon receptor (GLR) (23);
gastric inhibitory peptide receptor (GIPR) (24);
glucagon-related peptide receptor (GLPR) (25); growth
hormone-releasing factor receptor (GRFR) (26); receptor for
parathyroid hormone (PTRR) (27); diuretic hormone from tobacco hornworm Manduca sexta
(DIHR) (28); corticotropin-releasing factor receptor
(CRFR) (29); calcitonin receptor type A (CLRA) (30); and a hypothetical receptor from C. elegans, YOW3
(SwissProt P30650). Nonhomologous N- and C-terminal regions are not
shown. To optimize the alignment, dashes were introduced and
some short regions were omitted (dots). Residues identical
in more than 30% of proteins are shaded, and those
identical only in the "long receptors" are highlighted.
TMS positions are identified by arrows. Asterisks
mark conserved cysteines.
[View Larger Versions of these Images (64 + 99K GIF file)]
-cell lines. Consistent with its function as the LTX
receptor, latrophilin has only been found in those
-cell lines that
are responsive to LTX.2 Also,
a PC12 cell line that does not express latrophilin shows no toxin
binding and cannot be stimulated by
LTX.3
Fig. 3.
Identification of latrophilin as the
-latrotoxin receptor. A, Northern blot hybridization of
rat LPH1 mRNA (20 µg of RNA per lane). Lower panel,
control hybridization of 28 S RNA. B, time course of
expression of LTX receptors in COS cells transfected with
pcDNA3-LPH1 (
) or the vector alone (
). C,
Scatchard plot analysis of 125I-LTX binding to COS cells
transfected with LPH1. D, immunoblotting of latrophilin
expressed in transfected COS cells. Lane 1, latrophilin purified from rat brain, 10 ng; lanes 2, solubilized COS
cells transfected with the vector and enriched by WGA affinity
chromatography, 30 µg of protein; lanes 3, same as
lanes 2 but transfected with pcDNA3-LPH1. Latrophilin is
marked by an arrowhead.
[View Larger Version of this Image (37K GIF file)]
-subunit. We found
that when latrophilin purification is carried out in the presence of GDP and low micromolar Mg2+, a protein of 42 kDa
co-purifies with latrophilin, which reacts with antibodies against
G
o but not those against G
i subunits (Fig. 4, lane 1). Such
discrimination is likely to be specific because G
o
subunits are most abundant in neuronal plasma membranes, whereas
G
i subunits are present mainly on synaptic vesicles
(35). In addition, the preparation contained no trace of syntaxin,
SNAP-25, synaptotagmin, or synaptobrevin, proteins implicated in
vesicle docking/fusion (data not shown). The G protein band had been
absent from the receptor preparations made in millimolar
Mg2+ or in EDTA (12), pointing at physiological relevance
of this interaction. To further test the specificity of latrophilin
association with G
o, we subjected the preparation
containing both proteins to a second affinity chromatography using a
WGA column. As Fig. 4 (lanes 4-11) clearly shows, the two
proteins co-elute from the second column, indicating that their
interaction is stable during a two-step affinity purification. Our
results support the hypothesis that LPH1 is a GPCR; however, further
experiments are needed to unequivocally identify the specific
functional links and determine yet unknown effectors of
latrophilin.
Fig. 4.
Latrophilin interacts with a G protein.
A, latrotoxin (10 nM) stimulates cAMP and
IP3 production in COS cells transfected with LPH1 but not with
vector in the presence of 10 µM Ca2+.
B, solubilized rat brain was used for LTX affinity
chromatography in 20 µM Mg2+ and 2 mM GDP. The eluate from the LTX column (lane 1)
containing both latrophilin and a Go subunit was
subsequently loaded onto a WGA column (lane 2, flow
through), washed (lane 3), and eluted with 100 mM N-acetylglucoseamine (lanes
4-11). Fractions were analyzed by SDS-PAGE and immunoblotting
with respective antibodies. Note the absence of association between
latrophilin and G
i.
[View Larger Version of this Image (31K GIF file)]
*
This work is supported by a Wellcome Senior European
Research Fellowship (to Y. A. U.).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. Tel.:
44-171-594-5237; Fax: 44-171-594-5207; E-mail:
y.ushkaryov{at}ic.ac.uk.
1
The abbreviations used are: SV, synaptic
vesicles; GPCR, G protein-coupled receptor; IP3, inositol
1,4,5-triphosphate; LPH1, latrophilin 1; LTX, -latrotoxin; PAGE,
polyacrylamide gel electrophoresis; TMS, transmembrane segment; WGA,
wheat germ agglutinin; HPLC, high pressure liquid chromatography; kb,
kilobase pair(s).
2
J. Lang, Y. Ushkaryov, A. Grasso, and C. Wollheim, submitted for publication.
3
Y. Ushkaryov, unpublished data.
4
B. A. Davletov and Y. A. Ushkaryov, in
press.
Guyen, V. C., Roe, B. A., and Lipinski, M.
(1995)
Genomics
26,
334-344
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©1997 by The American Society for Biochemistry and Molecular Biology, Inc.