©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Site in the Substance P (NK-1) Receptor for Modulation of Peptide Binding by Sulfhydryl Reagents (*)

(Received for publication, October 12, 1995; and in revised form, November 10, 1995)

Hanzhong Li (1) Peggy Hsu (1) Bruce S. Sachais (2) James E. Krause (2) Susan E. Leeman (1) Norman D. Boyd (1)(§)

From the  (1)Department of Pharmacology, Boston University School of Medicine, Boston, Massachusetts 02118 and the (2)Department of Anatomy and Neurobiology, Washington University, School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The tachykinin peptide substance P (SP) (^1)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 alpha-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.


EXPERIMENTAL PROCEDURES

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 alpha-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 times 10^5 binding sites/cell) and C199S mutant (1.5 times 10^5 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(2) in alpha-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(2), 1 mM CaCl(2), 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(d) and B(max) 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.


RESULTS

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).


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 (bullet) and DTNB () and PCMB (box) 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.



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 (bullet) 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 I-(BH)-SP

The binding affinity at equilibrium (K(d)) and the maximum binding (B(max)) 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(d) of 0.4 nM and a B(max) of 1.2 times 10^5 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(d)) about 5-fold, without a significant change in the number of binding sites (Fig. 4A).


Figure 4: Effect of NEM on K, B(max), and the dissociation rate. Transfected CHO cells were pretreated at 4 °C for 1 h with 0.3 mM NEM (bullet) or buffer (circle) 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(max)) of 1.2 times 10^5 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).

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(n), 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).


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 (box), as well as NEM (bullet). 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.



Since the reactivity to the accessible sulfhydryl group was similar among the AM molecules, AM5 was selected as a representative. Its effect on K(d) 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(n) 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.

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 (bullet), C199S mutant receptor (box) (A) or AM5, wild type () and C199S mutant receptor (up triangle) (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 (bullet) or buffer (circle) 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.


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 nMI-(BH)-SP followed by addition of 1 µM unlabeled SP and 0.3 mM NEM (box), 10 mM AM5 (up triangle), or buffer alone (times). 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 (bullet) 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.




DISCUSSION

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(d) 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 beta-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(n)) 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 alpha-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 alpha-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 alpha 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.


FOOTNOTES

*
This work was supported by Public Health Service Grant NS31346 from the National Institute of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: L611/Dept. of Pharmacology, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-4387; Fax: 617-638-4374.

(^1)
The abbreviations and trivial names used are: SP, substance P; AM, 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.


ACKNOWLEDGEMENTS

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.


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