©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Arginine 206 of the C5a Receptor Is Critical for Ligand Recognition and Receptor Activation by C-terminal Hexapeptide Analogs (*)

Julie A. DeMartino (1) (5), Zenon D. Konteatis (2), Salvatore J. Siciliano (3), Gail Van Riper (1), Dennis J. Underwood (4), Paul A. Fischer (3), Martin S. Springer (3)(§)

From the (1)Departments of Biochemical and Molecular Pathology, (2)Analytical Biochemistry, (3)Immunology Research, and (4)Molecular Systems, Merck Research Laboratories, Rahway, New Jersey 07065 and the (5)Department of Molecular Genetics and Microbiology, UMDNJ, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

C5a is a 74-amino-acid glycoprotein whose receptor is a member of the rhodopsin superfamily. While antagonists have been generated to many of these receptors, similar efforts directed at family members whose natural ligands are proteins have met with little success. The recent development of hexapeptide analogs of C5a has allowed us to begin elucidation of the molecular events that lead to activation by combining a structure/activity study of the ligand with receptor mutagenesis. Removal of the hexapeptide's C-terminal arginine reduces affinity by 100-fold and eliminates the ability of the ligand to activate the receptor. Both the guanidino side chain and the free carboxyl of the arginine participate in the interaction. The guanidino group makes the energy-yielding contact with the receptor, while the free carboxylate negates ``electrostatic'' interference with Arg-206 of the receptor. It is the apparent movement Arg-206 induced by this set of interactions that is responsible for activation, since conversion of Arg-206 to alanine eliminates the agonist activity of the hexapeptides. Surprisingly, activation is a nearly energy-neutral event and may reflect the binding process rather than the final resting site of the ligand.


INTRODUCTION

The anaphylatoxin C5a is a 74-amino-acid glycoprotein generated on activation of the complement cascade. The responses evoked by C5a imply that it is an important mediator of inflammation(1, 2, 3) . For example, it is a potent chemotaxin and secretagogue for neutrophils, macrophages, eosinophils, and mast cells and stimulates the generation and release of histamine, prostaglandins, leukotrienes, interleukin-1, interleukin-6, and tumor necrosis factor. While C5a plays an important role in host defense, increased levels of C5a have been associated with a number of immune and inflammatory disorders including rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and acute respiratory distress syndrome(4, 5, 6, 7, 8, 9, 10) , observations that suggest that a C5a antagonist might have significant therapeutic utility.

The C5a receptor is a member of the rhodopsin superfamily(11, 12) , and while useful antagonists have been synthesized for many family members whose natural ligands are small molecules, similar efforts directed at family members that normally bind proteins have been largely unsuccessful. For example, although many moderately potent low molecular weight agonists have been developed for the C5a receptor, with one exception, attempts to develop antagonists have failed(13) . Thus, knowledge of the interactions between ligand and receptor is important not only for elucidating the mechanism of receptor activation but also for the design of antagonists.

The binding domain of the C5a receptor consists of two subsites(14, 15) . Site 1, located in the receptor's extracellular N terminus, interacts with the globular core of C5a, while site 2 binds the C-terminal 8 amino acids of C5a and appears to lie in the receptor's interhelical region. Site 1 provides the energy necessary for high affinity binding and also facilitates the interaction between the C terminus of C5a and site 2 of the receptor. It is this interaction at site 2 that is primarily responsible for receptor activation. Several laboratories have described hexapeptide analogs of C5a, which bind with moderate affinity at site 2(13, 16, 17) . Their small size makes these ligands readily amenable to modification and therefore useful probes for investigating the mechanisms of receptor activation. We have taken advantage of these properties and have combined a structure/activity study of the hexapeptide analogs with receptor mutagenesis to begin dissection of the molecular events that can lead to activation. In this communication we present evidence that movement of Arg-206 of the recep-tor, as a result of interaction with the ligands' C-terminal arginine, is a critical event in activation of the receptor by the hexapeptides.


EXPERIMENTAL PROCEDURES

Materials

Human C5a was purified and iodinated as previously reported(18) . Peptides were synthesized as reported previously, and their structures were confirmed by electrospray ionization mass spectrometry.

Receptor Mutagenesis and Transfection

Mutagenesis of the C5a receptor to replace Arg-206 with alanine was accomplished in two rounds of polymerase chain reaction, which overlapped and extended the receptor cDNA from the ScaI to NotI restriction sites. The R206A mutant cDNA expression plasmid was sequenced in its entirety prior to introduction of the plasmid into mammalian cells. Stable transfectants of the wild-type and mutant C5a receptors were generated as described previously(14) . To ensure that the phenotype of the R206A line is a reflection of the single mutation, and not of a single stable cell isolate, we demonstrated that binding properties of membranes prepared from HEK-293 cells transiently transfected with the R206A receptor were identical to those obtained from the stable line (data not shown).

Binding Assays

All assays were carried out by competition against I-C5a as described previously using membranes prepared from human neutrophils (19) or RBL-2H3 cells stably transfected with either the wild-type or R206A human C5a receptors (14). The buffer used for these assays was 0.05 M Hepes, pH 7.2, containing 0.1% bovine serum albumin, 5 mM MgCl, 1 mM CaCl, 1 mM phenylmethanesulfonyl fluoride, and 10 µg/ml each of aprotinin, chymostatin, and leupeptin.

Functional Assays

Measurement of myeloperoxidase release from human neutrophils (20) and ligand-induced Ca fluxes in transfected RBL-2H3 cells (14) were carried out as described previously.


RESULTS AND DISCUSSION

The Hexapeptide C-terminal Arginine Is Important for Both Receptor Activation and High Affinity Binding

Removal of the C-terminal arginine from C5a decreases its binding affinity for the wild-type receptor by 100-fold(21) , and as shown in , similar decreases also occur for the hexapeptide analogs, 1 and 2. Both of these analogs are agonists as measured by their ability to stimulate either degranulation (Fig. 1) or a Ca flux from human neutrophils, or a Ca flux from rat basophilic leukemia cells (RBL-2H3) stably transfected with the wild-type human C5a receptor(14) . However, these agonist activities are eliminated by removal of the C-terminal arginine as illustrated for degranulation in Fig. 1. The decrease in functional activity is not simply a reflection of decreased affinity since the EC values for peptides 1 and 2 are about 0.2 µM (EC degranulation/IC binding 3), while the des-Arg peptides show little activity at concentrations as high as 300 µM (EC/IC >30). Rather, the change represents a true loss of the ability to activate the receptor. Thus understanding the interaction of the C-terminal arginine with the receptor should provide insight into the activation process.


Figure 1: Removal of the C-terminal arginine reduces the agonist activity of the hexapeptides. The abilities of the peptides to activate the C5a receptor were evaluated by measuring the stimulated release of the granule enzyme myeloperoxidase (mpo) from human neutrophils. Data are shown for the agonists peptides 1 () and 2 () as well as des-Arg analogs 1d () and 2d () (see Table I for sequences). Also shown are the data for peptides 3 () and 4 () (see Table II for sequences). The results are the averages of triplicate determinations. Similar results were obtained from Ca flux experiments on both neutrophils and RBL-2H3 cells expressing the wild-type C5a receptor. Although not shown, peptide 5 (Table II) is an agonist as measured by both degranulation and Ca flux.



The Guanidino and Carboxyl Groups Are Both Necessary for High Affinity Binding

The hexapeptide's C-terminal arginine contains two functionalities that may participate in receptor binding, the side chain guanidino group and the free carboxylic acid. To probe the relative contributions of the two groups we have selectively eliminated them by making appropriate changes to the parent hexapeptide, 2. Substitution of agmatine for arginine, which deletes the carboxylate group while leaving the guanidino side chain, reduces affinity 200-fold to a value (IC = 10 µM) equivalent to that of the des-Arg peptide (2d and 3, ). Likewise, substitution of alanine for arginine, which deletes the side chain while leaving the carboxylate, reduces the affinity 40-fold (peptide 4, ). Thus, both the guanidino and carboxyl groups contribute to binding and appear to act in concert, as removal of either is equivalent or nearly equivalent to removal of the entire arginine residue.

Relationship between Arg-206 of the Receptor and the C-terminal Arginine of the Hexapeptides

The role the carboxyl group plays in interaction with the receptor is likely to be related to its electrostatic properties. This hypothesis is supported by the observation that amidation of the group, to eliminate the negative charge, produces a reduction in affinity similar to that caused by its deletion (peptide 5, ). Inspection of the receptor sequence suggested that Arg-206 at the top of transmembrane helix 5 (11, 12) was a likely candidate for interaction with the carboxyl group. Further, Arg-206 aligns with Lys-199 of the angiotensin II type 1 receptor, the residue proposed to interact with the carboxylic acid moiety of angiotensin II (22) and with Ser-203 of the -receptor, which appears to be involved in catechol binding(23) . Alanine substitution of Arg-206, to create the mutant C5a receptor R206A, eliminates the potentially interactive side chain while maintaining stereochemistry and allows us to test whether this is the site of contact.

If the C-terminal carboxylate group of the hexapeptide participates directly in an energy-yielding interaction with the guanidino group of Arg-206, removal of either group will eliminate that interaction resulting in a loss of binding energy. We have just shown that removing or amidating the peptide's C-terminal carboxylate results in a 40-200-fold loss of affinity against the wild-type receptor. A similar loss of affinity for carboxyl-containing peptides against the R206A receptor would be diagnostic of a positive interaction. However, the R206A mutation has little effect on the affinity of hexapeptides that terminate in arginine()or on C5a (), a result that clearly shows the lack of a positive interaction between the groups. Remarkably, however, peptides that contain a guanidino group but have their carboxyl groups deleted (peptide 3, ) or amidated (peptide 5, ) exhibit greatly increased affinity on the mutant receptor. In fact, the R206A mutation almost precisely compensates for the affinity loss caused by these carboxyl modifications when binding is measured against the wild-type receptor (). Thus, while there is little direct, energy-yielding interaction between the the peptides' C-terminal carboxyl group and the guanidino group of Arg-206, the R206A mutation eliminates the effect of, and the requirement for, the free carboxyl group. The role of the carboxyl group seems to be to prevent a loss of binding energy rather than as a positive contributor to binding.

These data strongly suggest that the basic side chain of Arg-206 is positioned so that it is an essential element of an electrostatic barrier to interaction between the receptor and the C-terminal guanidino moiety of the hexapeptides or C5a. The carboxylate group neutralizes the barrier so that repulsion is only apparent when ligands contain a positively charged C-terminal guanidino moiety but lack a corresponding negative charge normally supplied by the acid functionality. Under these conditions, Arg-206 impedes an energy-yielding contact between the guanidino group of the ligand and the wild-type receptor. Elimination of the barrier by removal of the side chain of Arg-206 restores potency to peptides with missing or blocked carboxyl groups. Ligands that contain a C-terminal arginine bind more avidly to the wild-type receptor because the negatively charged carboxylic acid moiety of arginine shields the opposing positive charges between ligand and receptor. Thus, Arg-206 functions as a gatekeeper denying access to positively charged ligands until presentation of a password in the form of a shielding negative charge.

Arg-206 Is Required for Receptor Activation by the Hexapeptides

Since the hexapeptide's C-terminal arginine is important for receptor activation and our data demonstrate an interaction between this group and Arg-206 of the receptor, it seemed likely that Arg-206 would play a key role in the activation process. This is indeed the case. As shown in Fig. 2, the agonist peptides 1 and 2 both elicit a Ca flux from RBL cells stably transfected with the wild-type receptor. In contrast, the same peptides fail to generate a Ca flux in cells transfected with R206A receptor, even at concentrations as high as 100 µM (Fig. 2). The inability to stimulate the mutant receptor is not due to global misfolding since the binding affinities of the two hexapeptides are unchanged by the arginine to alanine substitution. Similarly, hexapeptides 3 and 4, which are agonists on the wild-type receptor, fail to activate the R206A mutant. Thus Arg-206 plays an important role in receptor activation as well as ligand recognition for the hexapeptides. While the effects are not as dramatic, the R206A substitution alters activation by C5a as well. Greater concentrations of C5a are required to generate a Ca flux, and the profile of that flux has been changed by the mutation (Fig. 2).


Figure 2: The R206A mutation abolishes activation by hexapeptide agonists but not by C5a. The ability of the ligands to activate the receptors was determined by the induction of Ca fluxes in RBL-2H3 cells transfected with either the wild-type (A, C, E) or R206A (B, D, F) receptors. The hexapeptides were used at concentrations of 1 nM (), 10 nM (), 100 nM (), 1 µM (), 10 µM (), and 100 µM (▾). Results with the agonist hexapeptide 2 are shown in A and B and in C and D for hexapeptide 1. The effect of C5a, at concentrations of 1 pM (), 10 pM (), 100 pM (), 1 nM (), and 10 nM () are shown in E and F for the wild-type and mutant receptors, respectively. Both transfected lines expressed approximately 50,000 receptors/cell. Although not shown, peptides 3 and 4 also fail to activate the R206A mutant receptor. All of these hexapeptides are antagonists for the R206A receptor, since they block activation stimulated by C5a.



Model for Hexapeptide Activation of the Receptor

Our model for how the ligand recognition and activation functions of Arg-206 are coupled is shown in Fig. 3. At some point in the binding process the ligand's C-terminal arginine is located so that it is subject, either directly or indirectly, to substantial influence by Arg-206. This influence is bidirectional, altering the position of Arg-206 and initiating the conformational cascade that leads to receptor activation. More specifically, we believe that a transient electrostatic interaction between the C-terminal carboxylate of the ligand and the side chain of Arg-206 moves the latter, allowing the receptor to more readily accommodate the ligand's guanidino group. It is the interaction between this guanidino group and the receptor that accounts for most of the 2 kcal of binding energy provided by the ligand's C-terminal arginine. However, since the R206A substitution eliminates the ability of the arginine-containing hexapeptides to activate the receptor without altering their binding affinities, it is the movement of Arg-206 that is responsible for activation. Such a change in position might induce activation either because Arg-206, in its original location, acts as a negative regulator holding the receptor in a quiescent state or because its movement acts as a positive signal. Since the R206A receptor is not constitutively active, relocation of Arg-206 is a positive signal.


Figure 3: Model of receptor activation. Binding of the hexapeptides to the receptor involves an interaction between Arg-206 of the receptor and the ligand's C-terminal arginine. The free carboxyl of the C-terminal arginine negates electrostatic interference between the two guanidino groups, perhaps, as shown, by moving the side chain of Arg-206. It is the change in position of Arg-206 that appears to be responsible for hexapeptide-induced receptor activation. See text for details.



Although the details of how movement of Arg-206 leads to activation remain to be elucidated, two general classes of models can be proposed. First, Arg-206 may make a new set of specific interactions following ligand binding that initiate a conformational cascade in the receptor, or alternatively, the induced movement of Arg-206 may, somewhat less specifically, force a change in the relative orientations of the transmembrane helices. The observation that both peptide 3, which lacks the carboxyl group, and peptide 4, which lacks the guanidino group, can, unlike the des-Arg peptides, activate the receptor (Fig. 1) provides support for the second model. Although possible, it seems unlikely, that peptides 3 and 4 would force movement of Arg-206 to precisely the same location as the arginine containing peptide 2. However, both peptides 3 and 4 are likely to induce a change in the position of Arg-206, a movement that could alter the orientations of the helices. In this regard several groups have proposed such reorientation as the generic mechanism of activation for G protein-coupled receptors (24).

The activation pathway used by C5a also involves Arg-206, although the requirement is not absolute since C5a can induce functional responses in cells transfected with the R206A receptor. The mechanistic details that underlie the differences in activation pathways utilized by C5a and the hexapeptides remain to be elucidated, but they must reflect differences in the interaction domains between the receptor and the ligands.

Activation of the C5a receptor is an almost energy-neutral event. The R206A mutation has little effect on binding affinity but eliminates the ability of the hexapeptides to initiate activation. Similarly, a series of similar C5a hexapeptide analogs has been described in which changes in position 5 (C terminus is position 6) led to a progressive loss of agonism with little change in binding affinity(13) . This property may, in part, underlie the difficulties encountered in developing low molecular antagonists, as opposed to agonists, for this receptor(13, 16, 17, 25, 26) , since it suggests that the barrier to activation is low and readily overcome. However, most of those efforts have focused on maintaining, or mimicing, the interactions made by the C-terminal arginyl residue. While the one low molecular antagonist that has been described does contain a C-terminal arginine(13) , the current results suggest that such a strategy is more likely to generate agonism than antagonism and that a structure-based approach which avoids those contacts has a greater probability of success. Finally, our observations demonstrate that negation of deleterious interactions may be just as important as direct energy-yielding contacts in determining binding affinity and ligand-receptor specificity.

  
Table: Hexapeptides and the effect of removing the C-terminal arginine on affinity


  
Table: 1634494836p4in The IC of the peptides was determined by competition binding against I-C5a on membranes prepared from RBL-2H3 cells into which either the wild-type C5a receptor or the R206A receptor was stably transfected. The values shown are averages determined from the number of experiments given in parentheses.


FOOTNOTES

*
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: Dept. of Immunology, Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ 07065. Tel.: 908-594-4926; Fax: 908-594-7926.

In addition to the hexapeptides illustrated in Table II, the R206A mutation has little effect on the binding affinity of the hexapeptides described previously (13), including the antagonist, C089. All of these peptides terminate in arginine.


ACKNOWLEDGEMENTS

We thank Drs. Jerome Langer, William Moyle, and Linda Wicker for helpful discussions and critical reading of this manuscript.


REFERENCES
  1. Goldstein, I. M. (1988) in Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I., Goldstein, I. M., and Snyderman, R., eds) pp. 55-74, Raven Press, New York
  2. Hugli, T. E. (1984) Springer Semin. Immunopathol.7, 193-219 [Medline] [Order article via Infotrieve]
  3. Montz, H., Koch, K.-C., Zierz, R., and Gotze, O. (1991) Immunology74, 373-379 [Medline] [Order article via Infotrieve]
  4. Ward, P. A., and Zvaifler, N. J. (1971) J. Clin. Invest.50, 606-616 [Medline] [Order article via Infotrieve]
  5. Jose, P. J., Moss, I. K. Maini, R. M., and Williams, T. J. (1990) Ann. Rheum. Dis.49, 747-752 [Abstract]
  6. Hopkins, P., Belmont, H. M., Buyon, J., Phillips, M., Weissmann, G., and Abramson, S. B. (1988) Arthritis Rheum.31, 632-641 [Medline] [Order article via Infotrieve]
  7. Bergh, K., Iverson, O. J., and Lysvand, H. (1993) Arch. Dermatol. Res.285, 131-134 [Medline] [Order article via Infotrieve]
  8. Langlois, P. F., Gawryl, M. S., Zeller, J., and Lint, Y. (1989) Heart & Lung18, 71-84
  9. Gardinali, M., Padalino, P., Vesconi, S., Calcagno, A., Ciappellano, S., Conciato, L., Chiara, O., Agostoni, A., and Nespoli, A. (1992) Arch. Surg.127, 1219-1224 [Abstract]
  10. Takematsu, H., and Tagami, H. (1993) Arch. Dermatol.129, 74-80 [Abstract]
  11. Boulay, F., Mery, L., Tardif, M., Brouchon, L., and Vignais, P. (1991) Biochemistry30, 2993-2999 [Medline] [Order article via Infotrieve]
  12. Gerard, N. P., and Gerard, C. (1991) Nature349, 614-617 [CrossRef][Medline] [Order article via Infotrieve]
  13. Konteatis, Z., Siciliano, S. J., Van Riper, G., Molineaux, C. J., Pandya, S., Fischer, P., Rosen, H., Mumford, R. A., and Springer, M. S. (1994) J. Immunol.153, 4200-4205 [Abstract/Free Full Text]
  14. DeMartino, J. A., Van Riper, G., Siciliano, S. J., Molineaux, C. J., Konteatis, Z. D., Rosen, H., and Springer, M. S. (1994) J. Biol. Chem.269, 14446-14450 [Abstract/Free Full Text]
  15. Siciliano, S. J., Rollins, T. E., DeMartino, J., Konteatis, Z., Malkowitz, L., Van Riper, G., Bondy, S., Rosen, H., and Springer, M. S. (1994) Proc. Natl. Acad. Sci. U. S. A.91, 1214-1218 [Abstract]
  16. Kawai, M., Or, Y. S., Wiedman, P. E., Luly, J., and Moyer, M. (August 23, 1990) World Intellectual Property Organization, International Patent WO 90/09162
  17. Ember, J. A., Sanderson, S. D., Taylor, S. M., Kawahara, M., and Hugli, T. E. (1992) J. Immunol.148, 3165-3173 [Abstract/Free Full Text]
  18. Rollins, T. E., Siciliano, S., and Springer, M. S. (1988) J. Biol. Chem.263, 520-526 [Abstract/Free Full Text]
  19. Siciliano, S. J., Rollins, T. E., and Springer, M. S. (1990) J. Biol. Chem.265, 19568-19574 [Abstract/Free Full Text]
  20. Rollins, T. E., and Springer, M. S. (1985) J. Biol. Chem.260, 7157-7160 [Abstract/Free Full Text]
  21. Mollison, K. W., Mandecki, W., Zuiderweg, E. R. P., Fayer, L., Fey, T. A., Kranuse, R. A., Conway, R. G., Miller, L., Edalji, R. P., Shallcross, M. A., Lane, B., Fox, J. L., Greer, J., and Carter, G. W. (1989) Proc. Natl. Acad. Sci. U. S. A.86, 292-297 [Abstract]
  22. Yamano, Y., Ohyama, K., Chaki, S., Guo, D. F., and Inagami, T. (1992) Biochem. Biophys. Res. Commun.187, 1426-1431 [Medline] [Order article via Infotrieve]
  23. Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon, R. A. F. (1989) J. Biol. Chem.264, 13572-13578 [Abstract/Free Full Text]
  24. Underwood, D. J., Strader, C. D., Rivero, R., Patchett, A. A., Greenlee, W., and Prendergast, K. (1994) Chemistry & Biology1, 211-221
  25. Drapeau, G., Brochu, S., Godin, D., Levesque, L., Rioux, F., and Marceau, F. (1993) Biochem. Pharmacol.45, 1289-1299 [CrossRef][Medline] [Order article via Infotrieve]
  26. Or, Y. S., Clark, R. F., Lane, B., Mollison, K. W., Carter, G. W., and Luly, J. R. (1992) J. Med. Chem.35, 402-406 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.