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(Received for publication, October 12, 1995; and in revised form, November 10, 1995) From the
Substance P (SP) is a peptide neurotransmitter that is involved
in multiple responses in both the central and the peripheral nervous
systems through a G-protein-coupled receptor. The primary structure of
the rat SP receptor contains a number of conserved cysteine residues.
To localize and identify the cysteine residues that participate in
receptor binding, intact Chinese hamster ovary cells expressing the SP
receptor were treated with various sulfhydryl reagents and the effect
of these reagents on radioiodinated SP binding affinity and
dissociation rate was determined. We used a series of amphiphilic
maleimide derivatives in which the reactive maleimide group penetrates
to different depths within the plane of membrane. Only the maleimide
derivatives with intermediate chain lengths modified receptor binding
properties, indicating that the reactive sulfhydryl group is located
within a transmembrane domain of the receptor close (within 1.7 nm) to
the extracellular border. Since peptide binding to a mutant receptor
C199S, in which Cys-199 was replaced by a serine, was found to be
insensitive to modulation by sulfhydryl reagents, this reactive
sulfhydryl group is on Cys-199 of the receptor. Receptor occupancy by
SP protects Cys-199 from modification and thus this residue is either
located at or conformationally linked to the SP binding site.
The tachykinin peptide substance P (SP) ( In the absence of direct structural
information, chemical modification of functional groups is an useful
approach to investigate the relationship between structure and function
of receptor proteins. This approach is particularly informative if the
specific residue modified can be identified and its location with
respect to other structural features of the receptor protein including
the protein/lipid interface can be determined. The sulfhydryl group of
cysteine residues is distinguished by high chemical reactivity and by
the specificity of its chemical reactions. Reagents that modify
sulfhydryl groups and disulfide bonds have been used extensively to
study the structure and function of receptors in the G-protein-coupled
receptor superfamily including the photoreceptor rhodopsin(4) ,
opioid(5, 6) , muscarinic (7, 8) ,
dopaminergic(9, 10) , adenosine(11) ,
adrenergic(12, 13) , leukotriene B4(14) ,
vasopressin(15) , and vasoactive intestinal peptide
receptors(16) . Chemical modification of the SP receptor from
the rat brain membrane using disulfide bond reducing agents (17) and sulfhydryl reagents (18) has suggested that
peptide binding to and activation of SP receptors may also involve
disulfide bonds and sulfhydryl groups. Sequence analysis of the SP
receptor from various mammalian species, including
human(19, 20, 21, 22) ,
rat(23, 24) , mouse(25) , and guinea pig (26) , has shown that the receptor contains nine conserved
cysteine residues; two are located extracellularly, two are on the
intracellular carboxyl-terminal sequence, and five are contained within
the putative transmembrane spanning domains (Fig. 1). Recently
we provided evidence that in the rat SP receptor the two extracellular
cysteine residues (Cys-105 and Cys-180) are linked by a disulfide bond
that is an essential part of the SP binding pocket(27) . The
other cysteine residues do not appear to participate in disulfide bond
formation and thus, with the possible exception of cysteine 323, a
consensus site for
palmitoylation(28, 29, 30) , are potential
sites for modification by sulfhydryl-specific reagents.
Figure 1:
Topographical model
of the rat SP receptor showing seven putative transmembrane domains and
the locations of cysteine residues. Cysteines 105, 180, 199, 255, 260,
306, 307, 322, and 323 are conserved among all species cloned to date
and are indicated by dark circles. Cys-105 and Cys-180 are
shown linked by a disulfide bond(27) ; Cys-323 is a proposed
consensus site for palmitoylation(28) . Cys-80 is not present
in some mammalian species including human. Cys-199 is replaced by
serine in the mutant receptor C199S.
In the
present study, chemical modification and mutagenesis techniques have
been combined to identify the particular cysteine residue on the rat SP
receptor that is the target for modulation of binding affinity by
sulfhydryl-specific reagents. This approach has also allowed us to
determine the location of this cysteine residue within the plane of the
membrane and with respect to the peptide binding domain of the SP
receptor.
Figure 2:
Effect of sulfhydryl reagents on
The biphasic nature of the concentration
dependence curve for NEM suggested that there are at least two reactive
sulfhydryl groups of different sensitivity to NEM that are involved in
high affinity binding. The effect on SP binding by modification of the
more sensitive sulfhydryl group was studied in further detail. Prior to
the determination of the SP binding, intact transfected CHO cells were
treated with a low concentration of NEM at 0.3 mM, a
concentration that is sufficient to completely modify the more
sensitive site. The reduction in binding observed after the NEM
treatment was shown to be irreversible, since it persisted following
the removal of NEM by multiple dilution and cellular resuspension
steps. The time course of this reduction in SP binding produced by
treatment with 0.3 mM NEM was also measured. The decrease in
SP binding by NEM was rapid and even at 4 °C was complete within 10
min. No further reduction was observed up to 60 min (Fig. 3).
Figure 3:
Time
course of inhibition of
Figure 4:
Effect of NEM on K
The
dissociation rate of
Figure 5:
Effect of amphiphilic maleimide
derivatives on
Since the
reactivity to the accessible sulfhydryl group was similar among the AM
molecules, AM5 was selected as a representative. Its effect on K These results suggest that AM5 and NEM
produce similar effects on binding affinity and dissociation rates by
alkylation of the same residue. Since NEM can readily traverse the
cellular membrane, whereas AM
A
mutant rat SP receptor C199S in which Cys-199 was replaced with serine
by site-directed mutagenesis was expressed in CHO cells. This mutant
receptor has a slightly lower binding affinity and a higher
dissociation rate, characterized by values that are intermediate
between those of wild type receptors and the alkylated wild type
receptors. Of particular significance, no reduction in binding was
found following treatment of the C199S mutant receptor with AM5 for up
to 10 mM, as well as with NEM (Fig. 6). In addition,
the dissociation rate did not increase following treatment of the
mutant receptor with either AM5 (data not shown) or NEM (Fig. 7). These results confirmed that both sulfhydryl reagents
target the same sulfhydryl group and identified that this sulfhydryl
group is on Cys-199.
Figure 6:
Effect of AM5 and NEM on the binding of
mutant receptor C199S. A mutant rat SP receptor (C199S) in which
cysteine 199 was replaced with serine by site-directed mutagenesis was
expressed in CHO cells. Both the wild type and the mutant receptor were
pretreated at 4 °C for 1 h with the indicated concentrations of
either NEM, wild type (
Figure 7:
Effect of NEM on the dissociation rate of
Figure 8:
Effects of SP and CP 96,345 on alkylation
of cysteine 199 by AM5 or NEM. Transfected CHO cells were pretreated
for 1 h at 4 °C with either 100 nM unlabeled SP or 1
µM CP-96,345 or buffer alone (no ligand). At the end of
each pretreatment condition, NEM (0.3 mM), AM5 (10
mM), or buffer was added for an additional 1 h incubation. The
cells were then washed extensively, and the specific binding was
determined following equilibration with 0.5 nM
Figure 9:
The
dissociation rate of
Using a combination of chemical modification and
site-directed mutagenesis techniques, we have identified the sulfhydryl
group of cysteine 199 on the rat SP receptor as the site for modulation
of receptor binding affinity by sulfhydryl alkylating reagents. The
location of Cys-199 with respect to the extracellular lipid/water
interface was probed using a series of amphiphilic maleimide
derivatives. The results obtained established that this cysteine
residue is located in a transmembrane-spanning region of the SP
receptor, close to the extracellular phase boundary (within 1.7 nm).
Since the receptor occupancy by SP protected Cys-199 from modification,
this residue is at or conformationally linked to the peptide binding
site of the SP receptor. In an earlier study on the effect of NEM on
the peptide binding to SP receptors in rat brain membranes, it was
proposed that there are two different sulfhydryl groups that are
associated with peptide binding (18) . However, in that study
as well as many other similar studies on the effects of sulfhydryl
reagents on G-protein-coupled receptors, it was not possible to
identify sites of action involving cysteine on the receptor or on the
G-protein(5, 9, 10, 14) . In our
study, we observed about a 5-fold decrease in binding affinity after
NEM treatment at submillimolar concentration (Fig. 4A).
This would seem to rule out the involvement of G-protein inactivation
or uncoupling, since the SP receptor in the absence of G-proteins binds
SP with a K The chemical and physical properties of the amphiphilic
maleimide derivatives which we used to localize the cysteine residue on
the receptor have been described by Griffiths et
al.(34, 37) . As indicated by their partition
coefficients between octanol and phosphate buffer at pH 8, the
maleimide derivatives with a polymethylene chain of up to 10 carbons
are more soluble in the aqueous phase than in the lipid phase and their
respective carboxyl groups are ionized and will not penetrate the
membrane. They also share a similar reactivity toward
The binding
affinity of the SP receptor was shown to be decreased by pretreatment
with AM5 and AM7, but not by AM3 and AM10 (Fig. 5), suggesting
that the sensitive sulfhydryl group is located within the transmembrane
spanning domain at a distance between 1.1 and 1.7 nm from the
extracellular lipid/water boundary. This sulfhydryl group also appears
to be the site for modification by NEM, since binding to receptors
pretreated with AM5 was not reduced further following a second
treatment with NEM. The identity of the cysteine residue was
established by the finding that the binding of a mutant SP receptor,
C199S, is insensitive to modulation by either class of sulfhydryl
reagents. Moreover, treatment of C199S mutant receptor with either AM5
or submillimolar NEM did not produce a further decrease on receptor
affinity nor increase in the rate of dissociation (Fig. 6).
These results establish that the sulfhydryl group that is alkylated by
AM5, or by NEM, is on Cys-199 of the SP receptor. Hydropathic
analysis predicts that Cys-199 is located within the fifth
transmembrane-spanning region (TM V) close to the extracellular
lipid/water boundary. The present study supports the validity of the
hydropathic analysis and positions this residue about two There are two possible ways by
which receptor occupancy by the agonist shields the sulfhydryl group on
Cys-199 from alkylation: 1) direct shielding effect by the bound
agonist, and 2) an agonist induced conformational change of the
receptor protein. Current structural modeling of G-protein-coupled
receptors positions several or all of the seven TM-spanning helices
around an aqueous central cavity that penetrates deep within receptor
protein surface(3) . If Cys-199 is located within the central
core of the receptor, binding of SP could sterically block the access
of sulfhydryl reagents and thus protect Cys-199 from modification. This
residue would have to be in a hydrophobic pocket within the central
core since charged sulfhydryl reagents like PCMB, DTNB, and AM3 did not
modulate SP binding. Although an increase in chain length and thus in
hydrophobicity could explain the reactivity of maleimide derivatives
AM5 and AM7, it does not account for the lack of reactivity of the even
more hydrophobic derivative, AM10. A more feasible explanation is that
Cys-199 is located at the protein/lipid interface of the receptor, on
the opposite side of the The present study has demonstrated the value
of the membrane-impermeant sulfhydryl-specific reagents when used in
combination with site-directed mutagenesis to probe the
structure/function of the SP receptor. It should be possible to develop
other similar reagents with different chemical specificities as well as
to extend this approach to the other membrane-bound receptor proteins.
Volume 271,
Number 4,
Issue of January 26, 1996 pp. 1950-1956
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is widely
distributed in both the peripheral and central nervous systems and is
involved in multiple physiological responses including transmission of
painful stimuli and neurogenic inflammation(1) . These actions
are mediated by binding of the peptide to the SP receptor, which is
also termed NK-1 receptor. The cloning of SP receptor cDNA has allowed
the deduction of the primary structure of the receptor
protein(2) . Hydropathic analysis indicates that the SP
receptor is composed of seven hydrophobic segments of approximately 24
amino acids each, a characteristic property of G-protein-coupled
receptors. Based on sequence similarity with rhodopsin, whose structure
has been determined by two-dimensional electron crystallography, these
hydrophobic domains are presumed to form 
-helices that span the
cell membrane seven times(3) . However, it has not yet been
possible to directly study the structure of the SP receptor, due in
part to the difficulty in obtaining sufficient quantities of purified
receptor protein as well as an established method for crystallizing
membrane-bound proteins.
Materials
I-Labeled Bolton-Hunter
substance P (
I-(BH)-SP) was prepared as described
previously by Liang and Cascieri(31) . CP-96,345 was given by
Pfizer (Groton, CT). The N-polymethlyenecarboxymaleimides AM7
and AM10 were generously provided by Dr. Sidney Fleischer (Vanderbilt
University). AM5 (
-maleimidocaproic acid), AM3
(
-maleimidobutyric acid), N-ethylmaleimide (NEM),
5,5`-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid
(PCMB), SP, and
-modified minimum essential medium were purchased
from Sigma. Fetal bovine serum and Geneticin were from Life
Technologies, Inc., and enzyme free cell dissociation solution
(phosphate-buffered saline-based) was from Specialty Media (Lavallette,
NJ).Site-directed Mutagenesis, Stable Transfection, and Cell
Culture
Chinese hamster ovary (CHO) cells stably transfected
with the wild type (1.2 
10
binding sites/cell) and
C199S mutant (1.5 10 binding sites/cell) rat SP
receptor were generated by site-directed mutagenesis using the
M13-based oligonucleotide-directed in vitro mutagenesis system
from Amersham Corp.(32) . The oligonucleotide used was:
5`-AGTCACGCTGATGTG-3`. The sequence analysis of the point-mutated SP
receptor and stable transfection to CHO cells were described previously (32, 33) . The transfected CHO cells are maintained as
a monolayer culture at 37 °C in an atmosphere of 4% CO
in -modified minimum essential medium containing 10% fetal
bovine serum and 0.8 mg/ml Geneticin.Incubation with Sulfhydryl Reagents
Stably
transfected CHO cells were removed from the tissue culture dish with
the enzyme free cell dissociation solution, resuspended in HEPES buffer
(20 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.2
mM MgCl, 1 mM CaCl, 6 mg/ml
glucose, 0.6 mg/ml bovine serum albumin; pH = 7.4) containing
the sulfhydryl reagent at the indicated concentration, and incubated at
4 °C for 1 h. To determine the time course of the reduction in
binding, cells were pretreated with the sulfhydryl reagent for
different time periods followed by sedimentation, cell resuspension,
and subsequent assessment of binding.Receptor Binding Assay and Data Analysis
For
saturation binding, transfected CHO cells were incubated at 4 °C
for 1 h with increasing concentrations of I-(BH)-SP
(0.1-1 nM) to reach equilibrium. A mixture of protease
inhibitors: chymostatin (3 µg/ml), leupeptin (5 µg/ml), and
bacitracin (30 µg/ml), was added to prevent peptide degradation.
Binding was terminated by the addition of 5 ml of ice-cold HEPES buffer
and rapid filtering through a glass fiber filter (Whatman GF/C) that
had been soaked for at least 2 h in polyethylenimine (0.1%). The
filters were then assayed for radioactivity in a Nuclear Enterprises NE
1600
-counter at a counting efficiency of 70%. Nonspecific binding
was determined in the presence of excess unlabeled SP (1
µM). Binding data were analyzed by the non-linear
least-squares curve-fitting program in KaleidaGraph 3.0. Both K
and B
of I-(BH)-SP binding to the receptor were derived from the
Scatchard transformation.
Measurement of the Dissociation Rate
Transfected
CHO cells were equilibrated at 4 °C for 1 h with 0.5 nMI-(BH)-SP. The specific binding was measured at
increasing times following addition of excess unlabeled SP (1
µM).
Effect of Receptor Occupancy by SP and CP-96,345 on
Modification by Sulfhydryl Reagents
Transfected CHO cells were
pretreated at 4 °C for 1 h with 100 nM unlabeled SP, 1
µM CP 96,345, or buffer alone. At the end of each
pretreatment, NEM (0.3 mM), AM5 (10 mM), or buffer
was added for an additional 1 h incubation. The cells were washed as
described earlier, and the specific binding was then measured following
equilibration with 0.5 nMI-(BH)-SP.
Effect of Sulfhydryl-specific Reagents on Rat SP
Receptor Binding
The binding of I-(BH)-SP to
intact CHO cells expressing rat SP receptors was examined in the
presence of various sulfhydryl-specific reagents, NEM, DTNB, and PCMB.
Preincubation with NEM, which is uncharged and thus membrane-permeable,
reduced the specific binding of
I-(BH)-SP in a
concentration-dependent manner. In contrast, pretreatment with the two
charged reagents, DTNB and PCMB, produced little or no effect on the
receptor binding at concentrations as high as 10 mM (Fig. 2).
I-(BH)-SP binding to the rat SP receptor. Transfected CHO
cells were pretreated at 4 °C for 1 h with the indicated
concentrations of NEM (
) and DTNB () and PCMB (
)
prior to incubation with 0.5 nMI-(BH)-SP. The
specific
I-(BH)-SP binding was determined and is shown as
a percentage of the specific binding measured for cells pretreated with
buffer only. Results are the averages of duplicate determinations from
a representative experiment repeated at least three
times.
I-(BH)-SP binding by NEM.
Transfected CHO cells were pretreated at 4 °C with 0.3 mM NEM (
) for the indicated times and then assayed for specific I-(BH)-SP binding. The data are shown as percentages of
the specific binding measured for cells pretreated with buffer only.
Results are the averages of duplicate determinations from a
representative experiment repeated at least three
times.
Effect of NEM on the Binding Affinity and the Rate of
Dissociation of
The binding affinity at
equilibrium (KI-(BH)-SP
) and the maximum binding (B) of I-(BH)-SP to the rat SP
receptor were determined by Scatchard transformation of data derived
from saturation binding. The equilibrium binding of
I-(BH)-SP to the rat SP receptor was characterized by a K
of 0.4 nM and a B of 1.2 10 sites/cell (Fig. 4A). To examine the effects of alkylation of the
more sensitive sulfhydryl group on these equilibrium binding
parameters, binding to intact CHO cells transfected with SP receptors
that had been pretreated with 0.3 mM NEM for 60 min, was
measured. Scatchard analysis showed a decrease in the affinity of SP
binding (K) about 5-fold, without a significant
change in the number of binding sites (Fig. 4A).
, B
, and the
dissociation rate. Transfected CHO cells were pretreated at 4 °C
for 1 h with 0.3 mM NEM () or buffer (
) and washed
twice with buffer followed by equilibration
withI-(BH)-SP. A, equilibration at increasing
concentrations. Scatchard analysis of the data showed a binding
affinity (K
) of 0.4 nM and a
maximal binding (B
) of 1.2 10 sites/cell for the rat SP receptor. B, equilibration at
a fixed concentration of 0.5 nM. The dissociation rate was
measured by determining the specific binding of I-(BH)-SP
at indicated time following addition of excess unlabeled SP (1
µM). The inset shows a semilogarithmic plot of
the data. The data are presented as averages of duplicate
determinations of a representative experiment repeated at least three
times.
I-(BH)-SP bound to the SP receptor
was also examined by measuring the specific
I-(BH)-SP
binding at different time points after addition of excess unlabeled SP.
In the absence of NEM, the
I-(BH)-SP-receptor complexes
were quite stable at 4 °C and after 2 h following the addition of
the unlabeled SP only about 10% of the bound radiolabeled ligand had
dissociated from the receptor. Pretreatment with NEM prior to
equilibration with
I-(BH)-SP resulted in a marked
increase in the dissociation rate, which was now characterized by a t
= 60 min, representing at least an
8-fold increase in the dissociation rate (Fig. 4B).
This enhancement in the dissociation rate is in keeping with the nearly
5-fold decrease in binding affinity observed following submillimolar
NEM treatment (Fig. 4A).
The Location of the NEM-sensitive Sulfhydryl Group on the
Rat SP Receptor
The finding that the charged and thus
membrane-impermeant sulfhydryl-specific reagents, DTNB and PCMB, had no
effect on I-(BH)-SP binding to SP receptors on intact CHO
cells (Fig. 2) suggests that the cysteine residue that is
susceptible to alkylation by NEM is not located on the extracellular
surface of the SP receptor protein. To examine the possibility that the
cysteine residue is located on one of the seven putative TM-spanning
domains, we used a series of amphiphilic maleimide derivatives,
AM
, where n = 3, 5, 7, or 10 (see Fig. 5for structures). These N-polymethylenecarboxymaleimides consist of a negatively
charged carboxylate group separated from the reactive maleimide moiety
by increasing lengths of polymethylene chain. Pretreatment with either
the shortest chain or the longest chain maleimide derivatives, AM3 or
AM10, did not affect receptor binding; the derivatives with
intermediate chain lengths, AM5 and AM7, reduced the binding of I-(BH)-SP to an extent similar to NEM albeit at
concentrations about 10-fold higher (Fig. 5).
I-(BH)-SP binding. Transfected CHO cells
were pretreated at 4 °C for 1 h with increasing concentrations of
the amphiphilic maleimide derivatives, AM3 (
), AM5 (
),
AM7 (
), AM10 (
), as well as NEM (). Specific
binding was determined following equilibration with 0.5 nMI-(BH)-SP at 4 °C for 1 h. The inset shows the structure of amphiphilic maleimide derivatives and the
distance between the reactive double bond of the maleimide moiety and
the carbon atom of the carboxylate anion. The data are shown as
percentages of the specific binding measured for cells pretreated with
buffer only. Results are the averages of duplicate determinations from
a representative experiment repeated at least three
times.
and the dissociation was studied at 10
mM, a concentration that reduced the binding to the same level
as 0.3 mM NEM. A reduction in I-(BH)-SP binding
affinity to SP receptors by 10 mM AM5 was observed. This
decrease in binding affinity was paralleled by an enhanced dissociation
rate, which was similar to that produced by treatment with 0.3 mM NEM. In addition, when transfected CHO cells were treated with 10
mM AM5 followed by 0.3 mM NEM, there was no
additional effect by NEM.
molecules for n up
to 10 with charged carboxylate anion cannot(34) , the cysteine
residue that is sensitive to AM5, AM7, and NEM is located within the
plane of the membrane. Griffiths et al.(34) have
provided data that support a model for the mechanism of action of these
AM molecules, in which the carboxylate group anchors the reagents at
the lipid/water interface and the depths of the reactive maleimides can
reach within the plane of the membrane is dependent on the length of
their polymethylene chains. Thus, the cysteine residue sensitive to
AM5, AM7, and submillimolar NEM appears to be located within the
TM-spanning domain of the SP receptor close to the extracellular
border.Identification of the Reactive Cysteine Residue on the
Rat SP Receptor
As the proposed topographical model of the SP
receptor shows (Fig. 1), there are six cysteine residues that
lie within the seven putative TM regions of the receptor (cysteines 80,
199, 255, 260, 306, and 307). Of the six TM cysteine residues, Cys-80
on TM II and Cys-199 on TM V are located closest to the extracellular
border and are potential targets for alkylation by sulfhydryl reagents.
The possibility that Cys-80 is the target of NEM modification can be
eliminated since the human SP receptor, which has a serine residue in
place of Cys-80, is still sensitive to modification by AM5 or NEM.
), C199S mutant receptor () (A) or AM5, wild type () and C199S mutant receptor
(
) (B), followed by determination of specific I-(BH)-SP binding. The data are shown as percentages of
the specific binding measured for cells pretreated with buffer only.
Results are the averages of duplicate determinations from a
representative experiment repeated at least three
times.
I-(BH)-SP/C199S mutant receptor complex. Dissociation
rates of
I-(BH)-SP from C199S mutant receptor pretreated
with either 0.3 mM NEM (
) or buffer () of were
measured as described in Fig. 4B. The data are shown as
percentages of the specific binding measured for cells pretreated with
buffer only. Results are the averages of duplicate determinations from
a representative experiment repeated at least three
times.
Effect of Receptor Occupancy by SP and CP-96,345 on
Modification of Cys-199 by Sulfhydryl Reagents
In the absence of
added ligands, pretreatment with AM5 (10 mM) or NEM (0.3
mM) reduced the I-(BH)-SP binding by about 70%
of the control values in each case (Fig. 8). Incubation with a
saturation concentration of the non-peptide antagonist CP-96,345 (1
µM) (35, 36) prior to the addition of
either sulfhydryl reagents did not change the reduction in binding
produced by alkylation. In contrast, when the peptide agonist SP was
added prior to these sulfhydryl alkylating reagents, the reduction in
I-(BH)-SP binding was much less than that measured in the
absence of the peptide, suggesting that the binding of SP partially
protects Cys-199 from alkylation by AM5 or NEM (Fig. 8).
However, since the bound SP could not be completely washed away and
thus by itself caused a partial reduction in binding, it was difficult
to analyze the extend of protection provided by the peptide. To further
examine this question, we therefore designed a different protocol that
exploited the marked increase in the rate of dissociation which occurs
upon alkylation. The dissociation rate was determined in the absence or
presence of AM5 or NEM as the indicator of receptor modification by the
sulfhydryl reagents. When the receptor was occupied by
I-(BH)-SP, neither AM5 nor NEM had any effect on the rate
of dissociation when measured up to 2 h following addition of excess
unlabeled SP. This result is in marked contrast to the increased
dissociation rate that occurs within minutes of exposure of the
unoccupied receptors to these alkylating reagents (Fig. 9).
Taken together, these results indicate that when the SP receptor is
occupied by the agonist, the sulfhydryl group on Cys-199 is not
accessible to the sulfhydryl-specific reagents. By contrast, occupancy
of the SP receptor by the non-peptide antagonist CP-96,345 does not
protect against alkylation.
I-(BH)-SP. The data are shown as percentages of the
specific binding measured for cells pretreated with buffer only.
Results are the averages of duplicate determinations from a
representative experiment repeated at least three
times.
I-(BH)-SP/receptor complex in the
presence of sulfhydryl reagents. Transfected CHO cells were
equilibrated at 4 °C for 1 h with 0.5 nM
I-(BH)-SP followed by addition of 1 µM unlabeled SP and 0.3 mM NEM (
), 10 mM AM5 (), or buffer alone (). Specific binding of I-(BH)-SP was determined at indicated time. The data were
compared with the dissociation rate of the receptor pretreated with
either 0.3 mM NEM (
) or 10 mM AM5 ().
The data are shown as percentages of the specific binding measured for
cells pretreated with buffer only. Results are the averages of
duplicate determinations from a representative experiment repeated at
least three times.
about 30-50-fold greater. An
additional decrease in binding was observed at concentrations of NEM
greater than 10 mM. We have not yet explored the possibility
that the second NEM-sensitive cysteine residue is located on the
G-protein.
-mercaptoethanol. Furthermore, it has been demonstrated that all
the maleimide derivatives have equal potency in inhibiting the enzyme, D-3-phosphoglyceraldehyde dehydrogenase in the soluble state,
but varied in potency when the enzyme was embedded in the
membrane(34) . These amphiphilic maleimide derivatives have
been used to probe the depth of the sulfhydryl groups within the plane
of the membrane in several membrane-bound enzymes and transporter
proteins(34, 38, 39) . The results of these
studies provided strong support for a mechanism whereby the carboxyl
group anchors the maleimide derivatives at the membrane and the depth
of the maleimide group is determined by the length of their
polymethylene chains. The proposed penetration depths within the
membrane for these maleimide derivatives (AM) are defined
by the distance between the reactive double bond of the maleimide
moiety and the carbon atom of the carboxylate anion: 0.83 nm for AM3,
1.08 nm for AM5, 1.32 nm for AM7, and 1.71 nm for AM10.-helical
turns in depth (0.54 nm/turn) from the boundary. Results of the agonist
protection experiments suggest that Cys-199 is either located at or
conformationally linked to the peptide ligand binding pocket. The
location of the peptide binding pocket of SP has been investigated by
photoaffinity labeling and mutagenesis in previous
studies(27, 40, 41, 42) . These
studies have provided evidence that the second extracellular loop (E2)
of the rat SP receptor contains a contact site for SP. Therefore, it
may be of significance that Cys-199 located in TM V is close to the
point where E2 loop re-enters the membrane. In addition, TM V connects
the peptide binding domain with the third cytoplasmic loop (C3), which
is thought to be involved in G-protein coupling in the SP
receptor(28, 43) .-helix, away from the central core.
Following insertion into the membrane and lateral diffusion, maleimide
derivatives of only the appropriate length (AM5 and AM7) can alkylate
the sulfhydryl group on Cys-199. It seems unlikely that the binding of
SP to the E2 loop could directly block the access of sulfhydryl
reagents to Cys-199 at this proposed location. However, an
agonist-induced conformational transition of the receptor involving the
rotation of TM V helix could render Cys-199 no longer accessible
to sulfhydryl reagents. It seems reasonable that this membrane-spanning
sequence will play a key role in the conformation linkage of peptide
binding and G-protein activation. An allosteric interaction between
Cys-199 and the peptide binding site could also explain the decrease in
binding affinity that is associated with alkylation of Cys-199.
Moreover, the lack of a protective effect by the non-peptide antagonist
CP-96,345 is consistent with this latter model since binding of
antagonists will either produce different receptor conformational
changes from that of the agonists, or prevent agonist-induced
conformational changes.
), polymethylenecarboxymaleimide
with n methylene groups, where n = 3, 5, 7, or
10; CHO, Chinese hamster ovary; CP-96,345,
(2S,3S)cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-azabicyclo[2.2.2]octan-3-amine;
DTNB, 5,5`-dithiobis(2-nitrobenzoate); E2 loop, the second
extracellular loop;
I-(BH)-SP,
I-labeled
Bolton-Hunter substance P; NEM, N-ethylmaleimide; NK-1,
neurokinin-1; PCMB, p-chloromercuribenzoate; TM,
transmembrane.
We thank Dr. Sidney Fleischer for a gift of the
maleimide derivatives, AM7 and AM10, as well as Pfizer (Groton, CT) for
the SP antagonist CP-96,345.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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