(Received for publication, August 3, 1994; and in revised form, October 26, 1994)
From the
Glutamate receptors are the most abundant excitatory
neurotransmitter receptors in vertebrate brain. We have previously
cloned cDNAs encoding two homologous kainate receptors (GFKAR, 45
kDa, and GFKAR
, 41 kDa) from goldfish brain and proposed a
topology with three transmembrane domains (Wo, Z. G., and Oswald, R.
E.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7154-7158).
These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific
antibodies and point and deletion mutations. We report here that the
entire region between the previously proposed third and fourth
transmembrane segments is translocated and likely to be extracellular
in mature receptors. This was based on the following results. 1) The
entire segment was protected from Proteinase K and trypsin digestion
and could be immunoprecipitated by a site-specific antibody. 2)
Functional sites for N-glycosylation are present in the
C-terminal half of the segment, and 3) a mutation, constructed with an
additional consensus site for N-glycosylation in the
N-terminal half of the segment, was found to be glycosylated at that
site. Given the fact that the N terminus of the protein is likely to be
extracellular, this would place an even number of transmembrane
segments between the extracellular N terminus and the glycosylated
segment. In addition, results of N-glycosylation and
proteolysis protection assays of GFKAR
mutations indicated that
the previously proposed second transmembrane segment is not a true
transmembrane domain. These results provide further evidence in support
of a topology with three transmembrane domains that has important
implications for the relationship of structure to function in
ionotropic glutamate receptors.
Glutamate receptors are the primary excitatory neurotransmitter
receptors in the vertebrate central nervous system and are of
importance to a wide variety of normal and pathological processes. The
major forms of glutamate receptors are ligand-gated ion channels that
have been subdivided into AMPA()/kainate and NMDA receptors
according to their selective agonists(1) . Recent advances
achieved using molecular cloning and functional analysis have revealed
a high degree of molecular and functional complexity and
diversity(2, 3, 4) . Based on binding and the
activation of channel activity, the AMPA receptor subunits correspond
to GluR1 to GluR4, the kainate receptor subunits are GluR5 to GluR7 and
KA-1 to KA-2, and the NMDA receptor subunits are NMDAR1 and NMDAR2A to
NMDA2D. The individual subunits of these receptors are approximately
100 kDa. In addition to these functional ionotropic glutamate
receptors, lower molecular mass kainate subunits (40-50 kDa; also
referred to as kainate binding proteins) have been cloned from
nonmammalian vertebrate brain(5, 6, 7) .
These low molecular weight forms exhibit significant sequence
homologies with the C-terminal portions of the much larger 100-kDa
mammalian brain AMPA/kainate subunits. The sequence homology of these
40-50-kDa receptors is highest (
35-40%) with kainate
receptors (GluR5, GluR6, GluR7, KA-1, and KA-2 (2, 3) based on a BLAST (Basic Local Alignment Search
Tool) search(8) ). Although ion channel activity has yet to be
demonstrated in expression systems for these nonmammalian kainate
receptors, they have been implicated in the process of synapse
formation in the avian brain, as indicated by the finding that kainate
receptor expression is induced by an imprint stimulus in the duckling
hyperstriatum ventral(9) . Also, a recent report indicated that
the affinity column-purified lower molecular weight glutamate receptor
protein(s) from Xenopus brain can function as ion channels (10) . This suggests that the related kainate receptors from
other nonmammalian vertebrates may also be functional, but suitable
reconstitution and/or expression conditions to demonstrate functional
activity have not yet been found. The sequence homologies and similar
hydropathy plots suggest that their structures will be similar to the
C-terminal portion of the AMPA/kainate receptors(11) .
Therefore, determining the transmembrane topology of the
40-50-kDa kainate receptors should shed light on the structure of
the 100-kDa ionotropic glutamate receptors.
Ionotropic glutamate
receptors have long been assumed to belong to the superfamily of
ligand-gated ion channels(2, 4) , with nicotinic
acetylcholine receptors as the prototype. The proposed structural
features of this family are four putative transmembrane domains (TMs),
with both the N and C termini located extracellularly and with a large
cytoplasmic loop between the third (TMIII) and fourth (TMIV)
transmembrane domains(12) . In the case of glutamate receptors,
recent evidence suggests that the four-TM model may be incorrect. ()Like other ligand-gated ion channels, the N terminus of
glutamate receptors is likely to be located extracellularly since the
presence of the cleavable N-terminal signal sequence will translocate
the N-terminal region across the membrane of the endoplasmic reticulum
at the first step of the biosynthetic pathway (13) . This
notion is supported by the fact that, in some ionotropic glutamate
receptor (GluR) subtypes, potential N-glycosylation sites are
present in the N-terminal half of the protein, and these sites are
glycosylated(14, 15, 16) . Also, regions in
the N-terminal domain have been implicated in the binding of
agonist, polyamines, redox agents, and
zinc(17, 18, 19) . On the other hand, the
C-terminal tail of a subtype of the NMDA receptor (NMDAR1) was shown
using protein kinase C phosphorylation to be located
intracellularly(20) , which is consistent with the earlier
observation that the C-terminal tail of an NMDA receptor subunit
mediates the sensitivity of the response to the phorbol ester,
12-O-tetradecanoylphorbol-13-acetate (which activates
intracellular protein kinase C, 21) and that antibodies raised against
the C-terminal peptide stain the cytoplasmic side of cells expressing
rat brain AMPA/kainate
receptors(22, 23, 24) . These findings
indicate that ionotropic glutamate receptors have a unique topology.
The extracellular N terminus and cytoplasmic C terminus imply that an
odd number of transmembrane domains must be present, and models with
both three (6) and five (3, 20, 25) transmembrane domains have been
proposed.
Previously we reported (6) the cloning of two
goldfish kainate receptor subunits, GFKAR (45 kDa) and GFKAR
(41 kDa). The N-glycosylation of these two goldfish brain
kainate receptors was examined using a rabbit reticulocyte lysate in vitro translation/translocation system supplemented with
canine pancreatic microsomal membranes derived from endoplasmic
reticulum. Asn-307 of GFKAR
, an amino acid residue lying between
TMIII and TMIV, was found to be N-glycosylated. The site
homologous to Asn-307 in GluR6 (Asn-720) was also shown to be N-glycosylated(25, 26) . Therefore, at least
a portion of the region between the putative TMIII and TMIV, previously
thought to be cytoplasmic, must be extracellular. We showed further
that deletion of the putative TMII segment does not affect the N-glycosylation of GFKAR
, suggesting that this segment
may not be a true transmembrane domain. Given these results and
consideration of the hydrophobicity pattern, we proposed a model with
three transmembrane domains (TMI, TMIII, and TMIV) for goldfish kainate
receptors. On the other hand, to account for the reported
phosphorylation of GluR6 at Ser-684 by protein kinase
A(27, 28) , a five-TM model has been
proposed(3, 25, 26) . The N-glycosylation of Asn-720 of GluR6 constrains the possible
location of the proposed fifth TM segment between Ser-684 and Asn-720
in GluR6. As we noted previously(6) , this region (36 amino
acids) is hydrophilic, with numerous charged residues, and is,
therefore, not a likely candidate for a true transmembrane domain. The
sidedness of this region has become a key issue for solving the
transmembrane topology of ionotropic glutamate receptors.
In this
study, we have explored the transmembrane topology of GFKAR and
GFKAR
further using an in vitro translation/translocation
system. Two salient features of this system, N-glycosylation
and protection from protease digestion, were used in conjunction with
site/subunit-specific antibodies and point and deletion mutations. The
results are consistent with our previously proposed three-TM model (6) and specifically demonstrate 1) that the entire segment
between TMIII and TMIV is extracellular and 2) that only TMI, TMIII,
and TMIV are true membrane-spanning regions.
Figure 1:
Amino acid sequence alignment of
GFKAR and GFKAR
with rat 100-kDa ionotropic AMPA (GluR1) and
kainate receptor (GluR6) subunits in a key region between the proposed
TMIII and TMIV. Conserved residues are shown white on black. Consensus sites for N-glycosylation (g), Asn-307 of GFKAR
, and Asn-720 of GluR6, are in boldface. The reported phosphorylation site (p) of
GluR6, Ser-684, is shown in italic. E279GV of the
chimera was mutated to N279GT to introduce a consensus N-glycosylation site (g(new)) and is shadowed. The underlined sequences of GFKAR
and
GFKAR
were used to design the synthetic
peptides.
Binding of the site-specific antibodies to in vitro translated GFKAR and GFKAR
proteins was examined using
immunoprecipitation. We had reported previously that GFKAR
and
GFKAR
, translated in vitro, are 41-kDa proteins in the
absence of microsomal membranes. In the presence of microsomal
membranes, two N-glycosylated GFKAR
species of
approximately 45 kDa are routinely observed. The in vitro translation products from both subunits could be effectively
immunoprecipitated by the corresponding antipeptide antiserum.
Ab-
1 recognizes only the nonglycosylated and N-glycosylated GFKAR
(Fig. 2A, lanes1 and 2) but not GFKAR
(Fig. 2A, lanes5 and 6).
Ab-
1 recognizes only the translated GFKAR
in the absence and
presence of microsomal membranes (Fig. 2A, lanes9 and 10). No detectable translated products
were immunoprecipitated in the presence of corresponding peptide
immunogen (Fig. 2A, lanes3 and 4 for Ab-
1 and lanes11 and 12 for
Ab-
1).
Figure 2:
A,
immunoprecipitation of GFKAR and GFKAR
proteins translated in vitro in the absence or presence of microsomal membranes (M.M.). The asterisk indicates a glycosylated
species. Peptide immunogen refers to the addition of an excess of the
multiple antigen peptide peptide used in the production of the
corresponding antibodies. B, immunoblotting of GFKAR
(45
kDa) and GFKAR
(41 kDa) from goldfish brain (GF) and from
COS cells transiently expressing GFKAR
(C
) or
GFKAR
(C
). The asterisk indicates a
glycosylated species expressed in COS cells. Detectable signals are
seen with the corresponding antiserum (Ab-
1 or Ab-
1) diluted 1:3000.
The specificity of the two antipeptide antibodies for
GFKAR and GFKAR
was also established with an immunoblotting
assay. Membranes were prepared from goldfish brain and COS cells
transfected with GFKAR
and GFKAR
. When these membrane
proteins were probed with Ab-
1 and Ab-
1 antiserum (1:3000
dilution), specific immunoreactivity was detected. Ab-
1 recognizes
the 45-kDa protein in both goldfish brain membranes and
GFKAR
-transfected COS cells (Fig. 2B, lanes1 and 2). Ab-
1 recognizes the 41-kDa
protein in both goldfish brain membranes and GFKAR
-transfected COS
cells (Fig. 2B, lanes4 and 6). The doublet observed for transfected COS cells is probably
the result of different degrees of glycosylation because
under-glycosylation sometimes occurs in COS cells(35) . These
specific immunoreactivities were abolished using corresponding
competing peptide (Fig. 2B, lanes4-6 and 10-12). Thus, Ab-
1 and
Ab-
1 could be used selectively to identify the 45- and 41-kDa
kainate receptor subunits from goldfish brain, transfected COS cells,
and in vitro translation. We demonstrated, by comparison of
the protein sequence and DNA sequence(6, 32) , that
GFKAR
encodes the 41-kDa kainate receptor subunit and suggested
that GFKAR
is likely the cDNA for the 45-kDa kainate receptor
subunit from goldfish brain. The observation that Ab-
1 indeed
recognizes specifically a 45-kDa protein of goldfish brain membrane and
GFKAR
-transfected COS cells and that the 45-kDa protein is an N-glycoprotein both in vivo and in vitro further supports the assertion that GFKAR
encodes the
previously purified 45-kDa kainate receptor subunit from goldfish
brain.
Figure 3:
A, Synthesis and N-glycosylation
of native GFKAR and GFKAR
as well as GFKAR
(N307D). The
notation, g
-g
, denotes
0-3 oligosaccharide chains attached to the protein. B,
synthesis and N-glycosylation of GFKAR
, the
chimera, the
(N278GT) mutant, and the
(NKT439)
mutant in the presence and absence of microsomal membranes (MM). The consensus N-glycosylation sites, N278GT and
NKT439, were introduced to determine which would be glycosylated
(translocated). Endo H treatment removes attached oligosaccharide
chains. In B, asterisks indicate the glycosylated
species.
Figure 4:
A,
proteolysis and immunoprecipitation of native GFKAR and GFKAR
proteins and the
(N307D) mutant. Proteins were expressed in
vitro in the presence of microsomes. The translation mixtures were
either untreated or digested with Proteinase K in the absence or
presence of Triton X-100. Proteinase K-treated samples were divided for
treatment with Endo H as described under ``Experimental
Procedures.'' Samples were then immunoprecipitated with the
corresponding antiserum (Ab-
1 for GFKAR
and
(N307D) mutant, Ab-
1 for GFKAR
). The asterisk indicates a glycosylated species, and the arrow indicates
a deglycosylated species. B, proteolysis and
immunoprecipitation of native GFKAR
and the
(K186A)
mutant.
The above proteolytic patterns suggest that
the region between the TMI and TMIII may be the target of Proteinase K
and trypsin. Proteinase K primarily cleaves following the carboxyl
group of hydrophobic aliphatic and aromatic amino acids; whereas,
trypsin specifically catalyzes the hydrolysis of peptide bonds at the
carbonyl group of the basic amino acids, Arg and Lys. In GFKAR,
only three basic residues are present between TMI and TMIII: Arg-148,
Lys-186, and Arg-191. Arg-148 is located at the end of TMI and Arg-191
is present at the beginning of TMIII, placing them close to the surface
of the membrane and possibly not easily accessible to trypsin.
Therefore, we reasoned that Lys-186 could be a primary target for
trypsin and constructed a mutant to remove this potential site for
trypsin action. The
(K186A) mutant is expressed and glycosylated
in a manner indistinguishable from native GFKAR
. Also, proteolysis
by Proteinase K is identical to the wild-type protein (Fig. 4B, lane2versus6). In the case of trypsin, however, the pattern is
changed (Fig. 4B, lane3versus7). More than 50% of the protein is essentially intact
(approximately 42 kDa), with the remainder of the protein present as
two smaller bands (20 and 24 kDa). The most likely interpretation of
these results is that Lys-186 is the primary site of trypsin digestion
but that Arg-148 and Arg-191 may also be accessible to trypsin. This
would suggest that Lys-186 is present on the cytoplasmic side of the
membrane and that TMIII traverses the membrane from the intracellular
to the extracellular side.
Figure 5:
A, in vitro synthesis and N-glycosylation of
three GFKAR deletion mutations (
TMI,
TMII,
TMIII) in the presence or absence of microsomal membranes (MM). Endo H treatment removes attached oligosaccharide
chains. Positions of glycosylated subunits are indicated by asterisks. B, proteolysis (trypsin, Proteinase K) and
immunoprecipitation of the GFKAR
deletion mutations with
Ab-
1. Positions of the protected proteolytic fragments are
indicated by asterisks. C, schematics showing the
possible transmembrane arrangements of the deletion mutations
consistent with the N-glycosylation and proteolytic results.
Glycosylated consensus sites are indicated by
and those
consensus sites that are not glycosylated are shown by
.
Arrangements for which the epitope recognized by Ab-
1 is
proteolyzed are indicated by an opentriangle and
those for which the epitope is protected are shown with a filledtriangle.
We have extended our analysis of transmembrane topology of
the goldfish kainate receptors(6) . The two major conclusions
are that the 166-amino acid region between the proposed TMIII and TMIV
is entirely translocated across the endoplasmic reticulum membrane and
that the previously proposed TMII is not likely to be a transmembrane
helix. The first conclusion is based on the following observations. 1)
Three consensus sites for N-glycosylation are present in the
C-terminal half of this segment for GFKAR and one is present in
GFKAR
. All of these consensus sites are glycosylated. 2) A site
engineered into the N-terminal half of an
chimera (N279GT)
can also be N-glycosylated. 3) In an in vitro translation/translocation system, site-specific antibodies raised
against peptides in the middle of this segment can, following
proteolysis with Proteinase K or trypsin, immunoprecipitate N-glycosylated 24-30-kDa peptides. This would be the
size expected if the segment between TMIII and TMIV were entirely
translocated into the lumen of the endoplasmic reticulum and thus
protected from proteolysis. The second conclusion, concerning the
nature of TMII, is based on the following observations. 1) The finding
that the segment between TMIII and TMIV is extracellular would suggest
that an even number of transmembrane segments (probably 2 or 4) exists
between the extracellular N terminus and that segment. This would mean
either that one of the originally proposed TM segments does not
traverse the membrane or that an additional TM segment is present. 2)
Deletion of TMII has no effect on the N-glycosylation pattern,
suggesting that it is not a topogenic element, and 3) the deletion of
either TMI or TMIII affects the translocation of the region between
TMIII and TMIV and, thus, changes the N-glycosylation pattern.
4) Lys-186 of GFKAR
is the primary site of trypsin digestion.
These results lend further evidence in support of the three-TM model (Fig. 6), which we proposed previously(6) .
Figure 6:
Schematic depiction of the proposed
transmembrane topology (3-TM model) of GFKAR and GFKAR
. The
entire region between the TMIII and TMIV is on the extracellular side
of the plasma membrane because it is protected from protease digestion
and the four marked N-glycosylation sites are functional. The
three native consensus sites are indicated by
and the
site introduced by the mutation at position 279 is indicated by
. The sequence from which the antibodies were
raised is indicated with the boldfaceline and
indicated by Ab. We postulate that in vivo after
subunit folding and assembly of the receptor, the TMII region may be
associated with the ion channel pore but not necessarily in contact
with the membrane.
The
finding that the entire segment between TMIII and TMIV is extracellular
is not consistent with the reported phosphorylation of Ser-684 in
GluR6. In particular, the newly engineered consensus site, only 6 amino
acids C-terminal to the amino acid corresponding to Ser-684, was found
to be N-glycosylated. Raymond and co-workers (27) indicate that phosphorylation of Ser-648 alone is
sufficient to enhance responses to glutamate, whereas Wang and
co-workers (28) suggest that phosphorylation at both Ser-684
and Ser-666 is required. The reasons for the discrepancy between the
phosphorylation and glycosylation results are unclear; however, it is
noteworthy that in the case of the -amyloid precursor protein,
phosphorylation at an extracellular site in a transfected system has
been observed(41) . Also, the potential role of the consensus
protein kinase A sites in the intracellular C-terminal domain of GluR6
has not been investigated. Several lines of evidence, however, argue
against the intracellular location of Ser-684 in GluR6 and the
corresponding positions in GFKAR
and GFKAR
. 1) To account for
the N-glycosylation of Asn-720 (extracellular) and the
reported phosphorylation of Ser-684 (assumed to be cytoplasmic) of
GluR6, the proposed five-TM model must place a transmembrane domain
between Ser-684 and Asn-720(25, 26) . A
membrane-spanning domain in this portion of the protein is unlikely
because this region (35 amino acids) is very hydrophilic, with 7
charged amino acids. 2) The length of a transmembrane helix is
approximately 20 amino acids. If this region contains a true
transmembrane domain, Ser-684 and Asp-720 of GluR6 would be very close
to each side of the membrane surface. However, a minimum distance of
12-14 residues from the lumenal surface of the endoplasmic
reticulum membrane is required for efficient glycosylation (39) . 3) Transmembrane domains are important structural
components, and insertions or deletions in transmembrane domains have
not been observed within the same family, including other ligand-gated
ion channels(42, 43) . However, between the residues
corresponding to Ser-684 and Asn-720, a KG sequence in
GluR1-GluR4 is absent in GluR5-GluR7, KA-1 and -2, and the
40-50-kDa kainate receptors. The KG insertion is 16 residues
C-terminal to Ser-684 and 19 residues N-terminal to Asn-720. 4) At
least in the goldfish receptors, a mutation (
(N279GT)) that
creates a consensus site for N-glycosylation only 6 amino
acids C-terminal to the position corresponding to Ser-684 is in fact
glycosylated, suggesting that it is extracellularly located. Likewise,
the region just before TMIII is likely to be cytoplasmic based on the
(K186) mutant, which modifies the susceptibility to trypsin. Based
on these points, we propose that the entire region between TMIII and
TMIV is likely to be located on the extracellular side of the membrane.
The central reason for studying transmembrane topology is to provide insights into the structure of the receptors and the relationship between structure and function. Our proposed transmembrane topology (three-TM model) for ionotropic glutamate receptors is related to the formation of the channel pore, the intracellular regulatory sites, and the structure of the ligand binding site of this class of receptors. For other ligand-gated ion channels (e.g. the nicotinic acetylcholine receptor), significant divergence in sequence and length is observed in the cytoplasmic loop separating TMIII and TMIV. In contrast, this region is of fixed length and shows greater than 80% sequence identity among 100-kDa glutamate receptor subunits and 35% sequence identity between the 40-50-kDa kainate receptors and 100-kDa ionotropic glutamate receptors. This striking conservation unique to glutamate receptors indicates that this region may be structurally constrained. The residues in the N-terminal extracellular domain and in the loop between TMIII and TMIV are homologous to the Escherichia coli periplasmic glutamate binding protein(11, 44) ; and a two-domain glutamate binding site has been suggested by us and others (3, 6, 44, 45) . Interaction with the same endogenous ligand (glutamate) might be the structural constraint necessary to keep this region conserved. Strong support for this model comes from the analysis of the glycine binding site on the NMDA receptor, which showed that mutations both in the N-terminal extracellular domain and in the region between TMIII and TMIV can affect the action of glycine(45) .
Based on hydropathy
analysis (46) and presumed homologies to other ligand-gated ion
channels, cloned GluR subunits have been assumed, until recently, to
have five TM segments (i.e. the signal sequence plus four
membrane spanning regions found in the mature protein). Inspection of
the hydropathy plots for ionotropic glutamate receptor subunits in many
cases reveals only four regions of high hydrophobicity comprised of the
N-terminal signal sequence and three regions of sufficient length to
form -helical transmembrane domains (i.e. the proposed
TMI, TMIII, and TMIV). The proposed TMII in iGluRs is often
only weakly hydrophobic. In fact, as pointed out by Hollmann and
Heinemann(2) , a segment preceding TMI is as hydrophobic as the
proposed TMII. One can argue, based on the hydropathy analysis alone,
that: 1) both the TMII segment and the segment before the proposed TMI
act as transmembrane domains, or 2) neither are transmembrane domains
(3-TM model) because of their low hydrophobicity. Thus, no definitive
statement can be made without the benefit of additional experimental
data.
We have used an in vitro translation system to
analyze the transmembrane orientation of the polypeptide chain. In an in vitro translation system supplemented with microsomes,
previous results have shown that receptor subunits are synthesized,
inserted into the membrane, glycosylated, and undergo signal sequence
processing in a manner similar to that observed in mammalian
cells(47) . The transmembrane orientation assumed by the newly
synthesized subunits, at least for native proteins, is generally
identical to that of the subunits in the mature receptor complex. Where
tested, the pattern of glycosylation of GFKAR and GFKAR
that
we observe is the same in the in vitro translation system as
it is for the protein transiently expressed in COS cells. It has also
been demonstrated that, in the case of the nicotinic acetylcholine
receptor, the introduction of new glycosylation sites can provide
valuable information concerning the topographical location of
particular sites in the primary sequence of transmembrane proteins (48
Our use of the native GFKAR and GFKAR
proteins, in conjunction with immunoprecipitation, for the protease
protection experiments is a variant of the widely used reporter domain
technique for studying transmembrane topology(47) . The region
in GFKAR
and GFKAR
containing the sequence of synthetic
peptide to which antibodies were raised is, in effect, a reporter
domain specifically recognized by its corresponding antibody. In this
way, we avoided the concern that a foreign reporter domain might
disrupt the normal topology of the protein(49) , which is a
reasonable concern for artificial constructs (see below). Our three-TM
model places TMII on the cytoplasmic side of the membrane so that the
region between TMI and TMIII is not translocated and may be a target
for protease digestion. The observed patterns of Proteinase K and
trypsin digestion in GFKAR
and
TMII were consistent with
this arrangement. Both Proteinase K and trypsin digestion of GFKAR
gave a similar pattern of protected peptides, but
TMII is
resistant to Proteinase K and sensitive to trypsin. One possibility is
that TMII of GFKAR
is the target of Proteinase K digestion. When
it is deleted, the loop between TMI and TMIII is shortened to 28 amino
acids. This region is very hydrophilic, with 5 Arg/Lys residues, making
it susceptible to trypsin but possibly not Proteinase K. It may become
resistant to Proteinase K either due to the lack of hydrophobic
residues or potentially to the shortening in length, as has been
observed previously(47) . Inspection of the two short
hydrophilic segments flanking TMII may also yield clues to its
transmembrane orientation in that the surrounding charges can affect
the transmembrane behavior of a hydrophobic segment(50) . In
general, positive charges (Lys, Arg) tend to remain in nontranslocated
regions. Particularly for proteins with multiple transmembrane domains,
the charge bias is apparent for hydrophilic segments shorter than 70 or
80 residues(13) . The small polar segment (approximately 11
amino acids) between TMII and TMIII is positively charged, with 2 or 3
Arg or Lys residues. Both the four-TM and five-TM models place it
extracellularly (i.e. the models assume that it is
translocated). Our observation that TMII seems not to act as a true
membrane spanning region is consistent with the distribution of
charges. Perhaps, the combination of the low hydrophobicity and the
distribution of surrounding charges prevent TMII from functioning as a
true transmembrane domain in the coupled translation/translocation
step.
Unlike native proteins, deletion of the proposed TMI, TMII, or
TMIII from native GFKAR-generated artificial constructs. The
region carrying the previously identified functional N-glycosylation sites becomes the endogenous reporter domain,
and its membrane orientation, and thus N-glycosylation, is
determined by topogenic elements preceding it in the sequence.
Generally, the removal of upstream topogenic domains affects the
downstream region, whereas the removal of downstream topogenic elements
does not affect the topology of upstream regions(47) . Our
results showed that the region between the proposed TMIII and TMIV is
translocated (i.e. it corresponds to an extracellular domain);
therefore, an even number of transmembrane segments must exist between
this region and the N-terminal extracellular domain (either two- or
four-TM segments). Deletion of TMII does not affect the N-glycosylation; however, deletion of TMI or TMIII does affect
the N-glycosylation of the mutant proteins. This would suggest
that TMII may not be a true transmembrane segment in the native
proteins, but that TMI and TMIII are true transmembrane segments.
Nevertheless, the cases for
TMI and
TMIII are
somewhat complicated in that, particularly for
TMIII, a
mixture of forms was present (Fig. 5A, lane11). In general, topogenic sequences act in an absolute
manner, directing the nascent polypeptide to a single topologic form.
However, in a number of artificial protein constructs, topogenic
elements may act in a less than absolute fashion, producing more than
one topogenic form(51, 52, 53, 54) .
Thus, although the major species in each case was consistent with the
prediction of the three-TM model, alternative forms may have arisen
since the deletions produced artificial constructs.
Membrane-associated regions that arise from subsequent
rearrangements during subunit folding and assembly, however, may not be
detected in the above approaches. The P-segment of voltage-gated
K channels (55, 56, 57) and
several membrane segments (M5, M6, and M7) of the
H
,K
-ATPase (58) are such
examples. To account for its role in the ion permeation process, we
previously speculated that the proposed TMII, although not a true
transmembrane domain, may insert itself into the channel pore of the
receptor complex(6) . However, this type of ``membrane
domain'' is distinct from a true transmembrane-spanning segment in
the following ways. 1) The two types of structure arise differently.
True transmembrane domains of eukaryotic membrane proteins are inserted
in the coupled translation/translocation process. The insertion occurs
in a sequential fashion from the N-terminal to the
C-terminal(37) . A ``membrane domain'' such as the
P-segment in the K
channel arises from subunit folding
and assembly. It may not have any contact with the membrane lipid
bilayer at all. 2) True transmembrane-spanning regions are topogenic
elements that exert their effects on downstream regions of the
polypeptide. In contrast, the second type of ``membrane
domain'' has no topological effects on the sequences following it.
Therefore, identification of the second type of ``membrane
domain'' is an issue of protein folding/assembly rather than
translocation and transmembrane topology.
The RNA editing site (the
Gln/Arg site in the proposed TMII) suggests that the proposed TMII
segment is involved in the formation of the channel pore. This
postulated position of TMII brings the identified critical site for
channel properties (the Gln/Arg/Asn site) closer to the midpoint of the
channel. This position is more consistent with the measured electrical
distance (approximately 70% into the electric field from the
extracellular side) of the Mg blockade site (the Asn
in TMII) of NMDA receptors. It is difficult for the proposed four-TM
and five-TM models to accommodate this constraint(2) . The
outer mouth of the channel may be formed in part by the N-terminal
portion of the proposed TMI, as indicated by the finding that RNA
editing (generating Ile
Val and Tyr
Cys) of TMI of GluR6
also affects Ca
permeability(59) . These two
amino acid side chains modified by RNA editing would lie on the same
side of the transmembrane
-helix, exactly one turn apart. The
argument that ionotropic glutamate receptors have a distinct channel
pore structure not shared with nicotinic acetylcholine receptors is
also consistent with the finding that the serine residues in TMII of
GluR1 to GluR4 appear not to be involved in channel function, unlike
their role in nicotinic acetylcholine receptors(60) . Finally,
our three-TM model predicts that the C-terminal tail is located on the
cytoplasmic side, which is consistent with antibody staining of
AMPA-kainate receptors (22, 24, 61) and
phosphorylation of an NMDA receptor subunit(20, 21) .
The C-terminal tails of GluR6 and other kainate receptor subtypes are
rich in consensus protein kinase A sites, whether these sites have any
physiological role in protein kinase A-mediated phosphorylation of
GluR6 remains to be determined experimentally.
The fact that the ionotropic glutamate receptor family lacks sequence homology with other ligand-gated ion channels (i.e. those for which the nicotinic AChRs are the prototypes) and now seems to have a different transmembrane topology suggests that the glutamate receptor family may arise from different ancestral genes and consequently has its own unique structural design. Thus, ligand-gated ion channels very likely do not consist of a single superfamily of receptors. This is consistent with the recent cloning of an extracellular ATP-activated cation-selective ion channel, which defines a new family of ligand-gated ion channels with its pore-forming motif resembling that of potassium channels(62, 63) . The results described here are consistent with a three-TM model of transmembrane topology, but the structures of the ligand binding site, the channel pore, and the intracellular regulatory sites remain to be determined.