(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) ()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.
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 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 nM
I-(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.
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 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.
Figure 4:
Effect of NEM on K, 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
with
I-(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.
The
dissociation rate of 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).
Figure 5:
Effect of amphiphilic maleimide
derivatives on 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 nM
I-(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.
Since the
reactivity to the accessible sulfhydryl group was similar among the AM
molecules, AM5 was selected as a representative. Its effect on K 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.
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 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.
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 (), 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.
Figure 7:
Effect of NEM on the dissociation rate of 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.
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 nMI-(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.
Figure 9:
The
dissociation rate of 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.
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 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.
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
-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.
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 -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) .
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 -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.
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.