Tuftsin (An Ig-Associated Tetrapeptide) Triggers the Immunogenic Function

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Proc. Nati. Acad. Sd. USA Vol. 75, No. 7, pp. 34003404, July 1978 Immunology Tuftsin (an Ig-associated tetrapeptide) triggers the immunogenic function of macrophages: Implications for activation of programmed cells (tuftsin receptor/peptide hormones) E. TZEHOVAL*, S. SEGAL*, Y. STABINSKYt, M. FRIDKINt, Z. SPIRER*, AND M. FELDMAN* Departments of * Cell Biology and tOrganic Chemistry, The Weizmann Institute of Science, Rehovot, Israel; and * Pediatrics Department A, Municipal Covernmental Medical Center, Tel Aviv, Israel Communicated by Abraham White, April 24,1978

ABSTRACT The immunoglobulin heavy-chain-associated bradykinin (11) were synthesized as described. The "anti-al- tetrapeptide, tuftsin (Thr-Lys-Pro-Arg), known for its phago- lergic peptide," Asp-Ser-Asp-Pro-Arg, (12) was also synthesized cytosis-stimulating activity, was found to augment the antigen-specific, macrophage-dependent education of T lympho- in our laboratory (unpublished). Luteinizing hormone-releasing cytes.The investigation of stereospecific characteristics of the hormone (luliberin) was kindly donated by Ayerst Research tetrapeptide, by use of structural analogs with different modi- Laboratories, Montreal, Canada. Keyhole limpet hemocyanin fications, revealed strict structural requirements for eliciting (KLH) was purchased from Calbiochem (Venice, CA; lot 53006, the immunogenic activity of macrophages. It was found that A grade). 2-Mercaptoethanol was ordered from Eastman Kodak the most important moiety for its activity is the dipeptide Pro- Co., Rochester, NY. Dulbecco's modified Eagle's medium and Arg. Ths finding is of interest in view of the appearance of this Dulbecco's phosphate-buffered saline, pH 7.4, (Pi/NaCI) were particular dipeptide in other bioregulatory peptides, including supplied by the Cell Biology Department. RPMI 1640 (catalog many of the peptide hormones. The significance of the appearance of a commoil structure in such molecules, which may no. H-18) was purchased from GIBCO (Grand Island, NY). All act through specific receptors on different target cells, is dis- culture media were supplemented with penicillin (100 units/ cussed. ml), streptomycin (100 ,g/ml), and sodium bicarbonate (24 mM). Numerous studies have demonstrated the central role of T cells Preparation of Macrophages. BALB/c mice were injected in humoral and cellular immune responses. In addition to intraperitoneally with 3 ml of thioglycollate broth (Difoo). On functioning as effector cells, T cells may also augment as helpers the 5th day, the mice were killed by suffocation, with dry ice, or suppress the function of other cellular components of the and injected intraperitoneally with 10 ml of Pi/NaCl containing immune system (1). The activation by antigen of T cells, re- 5 units of heparin per ml (Thromboliquin N.V., Organon, Oss, sulting in antibody production, seems to depend on the presence Holland). The peritoneum was gently massaged and the peri- of macrophages (2-5). The presentation of antigen via the toneal fluid was aspirated with a 10-ml syringe. The cell sus- macrophage determines the triggering and generation of an pension was centrifuged in a refrigerated centrifuge at 200 X active T cell (4, 6). Yet, the mechanism that controls the acti- g for 10 min. The supernatant was discarded and the cells were vation of the immunogenic potential of the macrophages resuspended in Dulbecco's modified Eagle's medium, adjusted themselves is largely unknown. to a concentration of 2 X 106 peritoneal cells per ml. The sus- Studies of Najjar and his coworkers (7, 8) and those carried pensions contained approximately 90% macrophages and 10% out in our laboratory (9, 10)showed that the basic tetrapeptide lymphoid cells. L-threonyl-L-lysyl-L-prolyl-L-arginine, tuftsin, originating from In Vitro "Education" of Spleen Cells. In order to educate a specific leukophilic gamma globulin fraction, exhibits a high splenic lymphocytes, we used a method developed in our lab- activation potential for phagocytic cells: polymorphonuclear oratory and described elsewhere (13). In brief, 10 ml of mac- leukocytes (PMN) and macrophages. These experiments rophage suspension was plated on a 100-mm plastic tissue cul- demonstrated that tuftsin increases the phagocytic activity of ture petri dish (Falcon no. 3003, Falcon Plastics, Oxnard, CA). these cells (7-10) and potentiates several of their enzymatic The petri dishes were incubated overnight at 370 in a humi- systems involved in the pentose shunt (as assessed by en- dified atmosphere of 5% CO2 in air, to provide maximum ad- hancement of the nitroblue tetrazolium reduction test) (9, 10). herence of macrophages as a monolayer. The supernatant was led us to then discarded and the cultures were washed three times with These observations investigate possible stimulatory Pi/NaCl to remove most of the lymphocytes and to obtain as activity of tuftsin on the macrophage-dependent immune pure a population of macrophages as possible. To feed the "education" of T cells. The results suggest a general principle macrophages with antigen, we added 4 ml of Dulbecco's associated with the activating signal for certain classes of modified Eagle's medium containing 50 gg of KLH per ml to functionally programmed cells. each petri dish for 4 hr at 37°. In the experimental groups the MATERIALS AND METHODS medium also contained tuftsin or its analogs at various con- Mice. We used BALB/c female mice, 6-8 weeks old, sup- centrations. At the end of this incubation, excess free antigen plied by the Animal Breeding Center of the Weizmann Insti- was washed out by three sequential washes with an excess of tute. Pi/NaCl. At this stage, 10 ml of spleen cells (107 cells per ml) Chemicals and Media. Tuftsin and its analogs (9, 10, §) and with more than.90% viability (as assessed by the trypan blue dye The costs of publication of this article were defrayed in part by the Abbreviations: PMN, polymorphonuclear leukocytes; KLH, keyhole payment of page charges. This article must therefore be hereby marked limpet hemocyanin; Pi/NaCl, phosphate-buffered saline. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate § Stabinsky, Y., Fridkin, M., Zakuth, V. & Spirer, Z. (1977) Proceedings this fact. of the 44th Meeting, Israel Chemical Society, Abstr. MN-10. 3400 Downloaded by guest on September 29, 2021 Immunology: Tzehoval et al. Proc. Nati. Acad. Sci. USA 75 (1978) 3401

exclusion test) were added on top of the antigen-fed monmolyers and incubated at 370 in an atmosphere of 5% CO2 in air overnight. The nonadherent cells were then collected gently, re- seeded on another set of petri dishes (10 ml per dish), and incubated for 90 min to adsorb as many residual adherent cells as possible and thus minimize contamination with macrophages. The cells were then irradiated with 1000 rads, using a Der- mavolt x-ray machine (Siemens x-ray tube: 56 kV, 20 mA, 0.5 Al filter, at a dose rate of 750 rads/min). The suspension was collected, centrifuged, and washed twice at 250 X g for 10 min. The supernatants were discarded and the cells were resuspended in Eagle's medium and adjusted to a concentration of -400 108 nucleated cells per ml. Fifty microliters of this suspension were injected in the hind foot pads of syngeneic mice. On the 7th day, the popliteal lymph nodes were removed and a cell suspension was prepared in Pi/NaCl. The cells were washed 300- once and resuspended in culture medium (RPMI 1640 supplemented with 0.5% syngeneic mouse serum and 50 tiM 2- mercaptoethanol) to a concentration of 5 X 106 cells per ml. A 0. 1-ml sample of these cells was cultured in tissue microtiter 200 plates (Greiner, Nurtingen, Germany) in the presence of antigen or control reagents at 370 in a humidified atmosphere of 5% CO2 in air. After 72 hr, 2 of tritiated thymidine ,Ci (Israel 10O AEC, Negev, Israel) was added and the cells were allowed to incubate for 4 hr. Cells were then collected by a "Titertek" cell harvester (Skatronas, Lierbyen, Norway) on a glass filter, washed twice with saline, dried, and placed in Bray's scintillation fluid. Radioactivity was measured in a Packard Tri-Carb 0 20 160 liquid scintillation spectrometer. The results were expressed [TuftsinJ, nM in percent activation as to not compared controls treated with FIG. 1. Effect oftuftsin on augmentation ofimmunogenic activity tuftsin. ofantigen-fed macrophages. Macrophages were incubated with KLH and tuftsin at the indicated concentrations. Spleen cells were seeded RESULTS on those monolayers, irradiated, and injected in the hind foot pads. The in vitro antigen-specific response of 5 X 105 lymphocytes derived Guided by the fact that tuftsin augments the phagocytic activity from the popliteal lymph nodes to 0.5 Mg of KLH (0) or 0.05 Mg of of PMN and of macrophages, we designed our first experiment KLH (0) was determined by [3Hlthymidine incorporation. Results to test whether the peptide has the ability also to potentiate the are expressed as percent activation, compared to the control group immunogenic activity of macrophages. Macrophages were untreated with tuftsin and incubated with KLH alone. For experimental details, see legend to Fig. 2. incubated in the presence of various concentrations of tuftsin with a constant amount of antigen. Spleen cells were seeded as with that of some peptides unrelated to tuftsin. Such exper- for education on the macrophage monolayer and were then iments tested also whether there is a correlation between the injected into the hind foot pads of syngeneic mice. The popliteal tuftsin's capability to augment phagocytosis and its function lymph-node cells were then tested for response to antigen. The as an immunogenic stimulator. results (Fig. 1) indicate that tuftsin did produce a significant, concentration-dependent potentiation of the education capa- Table 1. Relationship of tuftsin-induced macrophage-mediated bility of antigen-fed macrophages. The activity of tuftsin had antigen presentation to sequence of exposure typical regulatory characteristics, with a maximum at about to antigen and tuftsin 160 nM per 20 X 106 macrophages. It was then important to establish a whether tuftsin is involved in general activation Group Treatment activation process of macrophages or whether it has to signal the macrophages concomitantly with the antigen, thus triggering an an- 1 Incubation of macrophages with KLH 100 tigen-dependent process. To test this, antigen was fed to mac- for 3 hr, preceded by 18-hr incubation rophages after preincubation of the macrophages with tuftsin without tuftsin over a period of 18 hr and washed to remove excess of tuftsin, 2 Incubation of macrophages with KLH 104 4 6 or simultaneously with tuftsin. The data presented in Table 1 for 3 hr, preceded by 18-hr incubation show that tuftsin acted only when presented at the time of an- with 50 1g of tuftsin tigen processing. It thus appears that the antigen's signal and 3 Incubation of macrophages with KLH 296 1 28 the signal of tuftsin have to act simultaneously to trigger the and 50 ;&g of tuftsin, preceded by immunogenic effect of macrophages. 18-hr incubation without tuftsin To test whether the activity of tuftsin is specific in its de- Macrophages were divided into three groups, which were treated pendence upon certain structural properties of the peptide or as indicated in the table. After washings, spleen cells were added on nonspecific and stemming, for example, from its relatively high top ofmacrophage monolayers, irradiated, and injected into the hind positive charge, we performed an additional set of experiments. footpads. The in vitro response of 5 X 10r popliteal lymph nodes to 0.5 of KLH was assayed by incorporation. The re- We tested the immunopotentiating effect of as com- ,sg [3H]thymidine tuftsin sponse of group 1 (7190 I 1179 cpm) was determined as 100% acti- pared with that of several structural synthetic analogs as well vation. Downloaded by guest on September 29, 2021 3402 Immunology: Tzehoval et al. Proc. Natl. Acad. Sci. USA 75 (1978) The results, summarized in Figs. 2-4, clearly demonstrate that the high stimulatory potency of tuftsin is specific and de- pends largely on the integrity of its amino acid sequence. Modification at either the NH2 or the COOH termini and within the chain generally leads to a reduction, abolishment, or even inhibition of macrophage activation. Certain structural alterations, however, do not significantly affect activity and may even lead to analogs with higher potency than tuftsin. Thus, the replacement of NH2-terminal Thr for Ser or its deletion led to analogs, [Serl]tuftsin and des-Thrl-tuftsin, re- spectively, with inhibitory activity. Substitution of Thr for Ala -300- on the other hand, yielded an analog, [Alal]tuftsin, with higher potency than tuftsin (Fig. 2). Masking of the positively charged guanidino function of Arg by a methyl group, as in [G-CH3- Arg4]tuftsin, reduced activity considerably. More pronounced 200- reduction and even inhibition resulted from neutralization of the positive charge by a nitro group or by substitution of Arg for Lys, as in [G-NO2-Arg4]tuftsin and [Lys4]tuftsin, respec- 100 tively. The latter peptide, however, exhibits a considerable tuftsin-like activity at high dosage (Fig. 3). Amidation of the COOH-terminal carboxylic group of Arg ([Arg-amide4]tuftsin) or replacement of L-Arg by D-Arg ([D-Arg4]tuftsin) hardly affected activity, whereas addition of Gly to the COOH ter- 0 10 20 70 80 90 100 minus (Thr-Lys-Pro-Arg-Gly) led to a significant increase in [Peptidel, nM activity (Fig. 3). Deletion of Arg-4 (Thr-Lys-Pro) resulted in FIG. 3. Effect of COOH-terminal analogs of tuftsin on the im- a marked reduction in activity, whereas deletion of Pro munogenic activity of antigen-fed macrophages. Macrophage or its substitution by Leu ([Leu3]tuftsin) abol- monolayers were incubated with KLH and [Arg-amide4]tuftsin, H- (Thr-Lys-Arg) Thr-Lys-Pro-Arg-CONH2 (0); or [D-Arg4Jtuftsin, H-Thr-Lys-Pro- ished activity (Fig. 4). D-Arg-OH (3); or [G-CH3-Arg4Jtuftsin H-Thr-Lys-Pro-G-CH3- Arg-OH (0); or [G-NO2-Arg4]tuftsin, H-Thr-Lys-Pro-G-NO2- Arg-OH (A); or [Lys4ituftsin, H-Thr-Lys-Pro-Lys-OH (A); or tuftsyl glycine, Thr-Lys-Pro-Arg-Gly (-), at the indicated concentrations. For experimental details see legend to Fig. 2.

500- Examination of the above results reveals that, with regard to tuftsin analogs, there is not always a correlation between the phagocytic and immunogenic effects of the peptides. Thus, for example, [Serl]tuftsin and des-Thrl-tuftsin also have a marked 400 inhibitory effect on phagocytosis of PMN (9, 10); [Leu3]tuftsin has a minor effect both on phagocytosing PMN (unpublished) and T-cell education. On the other hand, [Alal]tuftsin and 300- Thr-Lys-Pro-Arg-Gly exert opposite effects on the two cell functions. These peptides are pronounced activators of the macrophage-dependent T-cell education, while having prac- <200 tically no effect on phagocytosing PMN. Finally, (Lys)4, a highly positively charged tetrapeptide, has no stimulatory effect on phagocytosis by PMN, but it has an inhibitory effect on the 100 immunogenic function of macrophages (10). These data suggest that the signals for inducing immunogenic activity may be different from the signals for phagocytosis. Yet the apparent 160 170 180 190 200 difference between immunogenic and phagocytic reactivities 0 10 20 may actually reflect differences between receptors of macro- [Peptide], nM phages and of PMN or between mouse and human PMN. FIG. 2. Effect of NH2-terminal analogs of tuftsin on the immunogenic activity of antigen-fed macrophages. Macrophage monolayers The structural requirements of tuftsin for the manifestation were incubated with KLH and [Alal]tuftsin, H-Ala-Lys-Pro-Arg-OH of macrophage stimulation suggest the existence of a specific (0); or [SerlJtuftsin, H-Ser-Lys-Pro-Arg-OH (-); or des-Thrl-tuftsin, receptor for the regulatory peptide. Further examination of the H-Lys-Pro-Arg-OH (A) at the indicated concentrations. Spleen cells results depicted in Fig. 4 may shed light on the nature of this were added on top of the macrophages, irradiated, and injected into receptor. They indicate that the amino acid couple Pro-Arg is the hind foot pads. The in vitro antigen-specific response of 5 X 105 probably the dominating obligatory entity recognized by the popliteal lymph-node lymphocytes to 0.5 ,g of KLH was assayed by of of the [3H]thymidine incorporation. Results are expressed as percent acti- receptor. Indeed, assessment the stimulating capacity vation compared to the control group incubated with KLH alone. In dipeptide prolyl-arginine on macrophages revealed a significant this experiment, as well as in the former and subsequent experiments, potency (Fig. 4). Furthermore, evaluation of macrophage ac- 100% activation in the presence of 0.5 ,g of KLH per culture corre- tivation by three naturally occurring peptides that are not re- sponds to 11,758 4 1338 cpm over background of antigen-unstimu- lated to tuftsin but contain the sequence Pro-Arg or the retro- lated cultures. The range oftuftsin concentrations used in all exper- sequence Arg-Pro gave similar results. The peptides studied iments was based on numerous previous titrations of tuftsin, its an- Pro- alogs, and other peptides. Each point was determined by three rep- were Hamburger's "anti-allergic peptide," Asp-Ser-Asp licate cultures. Arg (12), bradykinin, and leuteinizing hormone-releasing Downloaded by guest on September 29, 2021 Immunology: Tzehoval et al. Proc. Nati. Acad. Sci. USA 75 (1978) 3403

&> 300

200

0 20 60 80 100 120 140 [Peptide], nM FIG. 4. Effect of peptides with partial tuftsin sequences and of [Leu31tuftsin on the immunogenic activity of antigen-fed macrophages. Macrophage monolayers were incubated with KLH and Thr-Lys-Arg (-); or Thr-Lys-Pro (a); or Pro-Arg (0); or [Leu3]- 0 10 20 60 70 80 tuftsin, H-Thr-Lys-Leu-Arg-OH (@) at the indicated concentrations. [PeptideJ, nM For experimental details see legend to Fig. 2. FIG. 5. Effect of peptides unrelated to tuftsin on the immunogenic activity ofantigen-fed macrophages. Macrophage monolayers hormone (luliberin) (the two latter peptides possessing an were incubated with KLH and "anti-allergic peptide," H-Asp-Ser- Arg-Pro sequence). As shown in Fig. 5, all three peptides, and Asp-Pro-Arg-OH (0); or luteinizing hormone-releasing hormone, particularly the first one, are active, though in higher molar < Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-CONH2 (0); or concentrations than tuftsin. bradykinin, H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH (M); or Although we suggest the function of membrane-associated (Lys)4, H-Lys-Lys-Lys-Lys-OH (-) at the indicated concentrations. cell receptor for tuftsin, alternative sites of interactions re- For experimental details see legend to Fig. 2. quiring structural specificities could not, at this stage, be ex- by nonspecific enzymes (e.g., see ref. 14). These results, in ad- cluded. dition to previous reports describing the PMN-activating properties of tuftsin (7-10), clearly establish the existence of DISCUSSION an Ig-bound peptide, tuftsin, that potentiates the immunogenic In the present study we demonstrated that the phagocytosis- function of the mammalian macrophages, resulting in an ef- stimulating peptide, tuftsin, has a definite ability to potentiate fective education of the T cell. Even though opsonic activity the antigen-specific "educating" capacity of macrophages. This of certain isotypes of antibodies is well known, we still do not potentiating activity is specific for the intact sequence of tuftsin. understand whether this interaction of antigen-antibody Comparative structural analysis of several of its synthetic an- complexes with the surface of macrophages is merely an af- alogs indicates the possible existence of a specific receptor entity finity-increasing device or whether it also serves specifically for this peptide, which is probably dominated by the Pro-Arg to induce events that cause the immunogenic processing of the sequence. This assumption is further supported by the finding engulfed antigen. Our data strongly support the latter as- that the dipeptide prolyl-arginine as well as the "anti-allergic sumption. Since tuftsin is part of the constant region of the Fc peptide" has a considerable stimulatory action. The Pro-Lys portion of the Ig molecule, the understanding ot its mode of sequence, in which a similarly positively charged amino acid action may elucidate the function of the Fc portion and its is located next to proline, is probably also recognized by the regulatory role. It is of interest to reveal the results described receptor, as can be inferred from the tuftsin-like activities of in Fig. 5, in which it is shown that the "anti-allergic peptide" Thr-Lys-Pro-Lys (Fig. 3). The receptor has also some affinity (12), which is located in the CH2 region of the IgE heavy chain to the reverse sequence Arg-Pro. This is deduced from the ac- and which acts through a specific receptor on a very closely tivity of bradykinin and of luteinizing hormone-releasing related cell, the basophilic PMN, is also active to a significant hormone (Fig. 5). It seems plausible that the specific binding level on the peritoneal macrophages. Tuftsin, on the other hand, of the amino acid couple, Pro-Arg, and to a lesser extent, Pro- slightly but distinctly inhibited the degranulation of mast cells Lys, and to an even lesser extent, Arg-Pro, stems from the fact (unpublished), i.e., it exerted an "anti-allergic-peptide"-like that positively charged amino acid is forced by Pro into a activity. This finding supports the concept of a related and negative locus within the receptor site. As an amino acid, Pro conservative evolution of the Fc of the Ig molecule and the is unique in its formation of a secondary amide peptide bond, specific cellular receptors for Ig, and strengthens the possible which has particular structural-conformational implications. regulatory role of the Pro-Arg-associated sequence of the CH2 In addition to its structural role, the Pro may determine the region of the Ig molecule. protection of the peptide's active epitope against degradation The ability of peptides and peptide hormones unrelated to Downloaded by guest on September 29, 2021 3404 Immunology: Tzehoval et al. Proc. Natl. Acad. Sci. USA 75 (1978) Table 2. Occurrence of Pro-Arg, Pro-Lys, and Arg-Pro sequences regulatory peptides is also strongly supported by the extensive in some biologically active peptides (25) studies of Chipens et al. (26). Peptide Sequence M. Feldman is an established investigator of the Bureau of the Chief Arginine-vasopressin -Pro-Arg-Gly-NH2 of "Anti-allergic-peptide" Scientist of the Israel Ministry Health. (13) H-Asp-Ser-Asp-Pro-Arg-OH Neurotensin -Lys-Pro-Arg-Arg-Pro- 1. Cantor, H. & Boyse, E. A. (1977) Cold Spring Harbor Symp. Adrenocorticotropin Quant. Biol. 41, 23-32. hormone -Arg-Arg-Pro-Val- 2. Unanue, E. R. (1972) in Advances in Immunology, eds. Dixon, Luteinizing hormone- F. J. & Kunkel, H. G. (Academic, New York), Vol. 15, pp. 95- releasing hormone -Leu-Arg-Pro-Gly-NH2 165. Substance P H-Arg-Pro-Lys-Pro- 3. Ishizaka, K. & Adachi, T. (1976) J. Immunol. 117,40-47. Bradykinin H-Arg-Pro--- Pro-Phe- 4. Thomas, D. W. & Shevach, E. M. (1976) J. Exp. Med. 144, Arg-OH 1263-1273. Lysine-vasopressin -Pro-Lys-Gly-NH2 5. Thomas, D. W. & Shevach, E. M. (1977) Proc. Natl. Acad. Sci. Melanocyte stimulating USA 74,2104-2108. hormone -Pro-Pro-Lys- 6. Unanue, E. R. & Askonas, B. A. (1968) J. Exp. Med. 127,915- Insulin B-chain, human -Pro-Lys-Thr-OH 926. 7. Najjar, V. A. & Nishioka, K. (1970) Nature 228,672-673. 8. Nishioka, K., Constantopoulos, A., Satoh, P. S. & Najjar, V. A. tuftsin but carrying either the Pro-Arg or the Arg-Pro constit- (1972) Biochem. Biophys. Res. Commun. 47,172-179. uent to stimulate macrophage and PMN is of particular interest. 9. Fridkin, M., Stabinsky, Y., Zakuth, V. & Spirer, Z. (1976) in Comparing the primary structures of various regulatory hor- Peptides 1976, Proceedings of the 14th European Peptide mones, we were impressed by the common abundance of these Symposium, ed. Loffet, A. (Presse Universitaire de Bruxelles, sequences (Table 2). Several questions can be raised. For ex- Bruxelles, Belgium), pp. 541-550. ample: Does the "insulin-like" activity of vasopressin (15, 16) 10. Fridkin, M., Stabinsky, Y., Zakuth, V. & Spirer, Z. (1977)BPochim. stem from the Pro-Arg sequence which corresponds to Pro-Lys Biophys. Acta 496, 203-211. (residues 28-29) of the B chain of insulin? Can the Pro-Arg- 11. Fridkin, M., Patchornik, A. & Katcalski, F. (1968)J. Am. Chem. Arg-Pro- sequence of neurotensin account for its wide spectrum Soc. 90,2953-2957. of activity, e.g., the elevation of plasma gonadotropin levels, 12. Hamburger, R. N. (1975) Science 189,389-390. the luliberin-like activity (17), or the kinin-family activity (18)? 13. Steinman, L., Tzehoval, E., Cohen, I. R., Segal, S. & Glickman, Why does the "contraceptive" peptide Thr-Pro-Arg-Lys exhibit E. (1978) Eur. J. Immunol. 8,29-34. some luteinizing hormone-releasing hormone-like activity 14. Yaron, A. (1974) Israel J. Chem. 13,651-662. (19)? 15. Braun, T., Hechter, 0. & Rudinger, J. (1969) Endocrinology 85, We would like to suggest a possible hypothesis, which can 1092-1096. 16. Bonne, D. & Cohen, P. (1975) in Peptides: Chemistry, Structure as 20 be summarized follows: Numerous studies (e.g., refs. and and Biology, Proceedings of the 4th American Peptide Sympo- 21) have revealed the existence of specific membrane-bound sium, eds. Walter, R. & Meienhofer, J. (Ann Arbor Science receptors which, by their interactions with various regulatory Publishers, Ann Arbor, MI), pp. 711-717. molecules, will lead to a variety of intracellular events, resulting 17. Makino, T., Carraway, R., Leeman, S. & Greep, R. 0. (1973) Biol. in the expression of the cell's programmed state. It is logical to of Reproduction 9,66. postulate that such central events, which are triggered through 18. Carraway, R. and Leeman, S. E. (1973) J. Biol. Chem. 248, signals received by different receptor systems, should end in 6854-6861. activation via a common dominant end signal. We suggest, 19. Chang, D., Griebrokk, T., Knudsen, R., Howard, G., Humphries, therefore, that this common end signal is, at least for some target J., Folkers, K. & Bowers, C. Y. (1975) Biochem. Blophys. Res. cells, activated by the Pro-Arg sequence, which acts as a dom- Commun. 65,1208-1213. inant epitope of the regulatory molecule, while the accompa- 20. Levy, D. (1973) Biochim. Blophys. Acta 322,329-3. nying neighboring amino acids determine the affinity and 21. Cuatrecasas, P. & Hollenberg, M. D. (1976) Adv. Protein Chem. specificity for the particular target cells. This assumption gains 30,252-450. support from three recent studies (22-24) that analyzed the 22. Seelig, S., Sayers, G., Schwyzer, R. & Schiller, P. (1971) FEBS binding of segments as well as of intact adrenocorticotropin Lett. 19, 232-234. hormone and of neurotensin (24). They suggested that Arg-Pro 23. Hofmann, K., Wingender, W. & Finn, F. M. (1970) Proc. Natl. Acad. Sci. USA 67,829-836. and Pro-Arg determinants are directly associated with the to their 24. Carraway, R. & Leeman, S. (1975) in Peptides: Chemistry, binding and activity of the hormones with respect Structure and Biology, Proceedings of the 4th American Peptide various cellular receptors. Symposium, eds Walter, R. & Meienhofer, J. (Ann Arbor Science Finally, one may wonder whether the relatively high fre- Publishers, Ann Arbor, MI), pp. 679-685. quency of the sequences Pro-Arg or Pro-Lys (and Arg-Pro) is 25. Dayhoff, M. 0. (1972) Atlas ofProtein Sequence and Structure, indeed limited only to a category of regulatory peptides, or (National Biomedical Research Foundation, Washington, may, according to some probability considerations, be found DC). in similar abundance in many other proteins. The fact is, 26. Chipens, G. I., Auna, Z. P., Klusha, V. E.,Krikis, A. J., Pavan, A. however, that a large number of other analyzed polypeptides, P., Papsucrich, 0. S., Romanovsk P. J. & Vegner, R. E. (1973) especially enzyme molecules, lack these particular dipeptide in Peptides 1972, ed. Hanson, H. (North-Holland, Amsterdam, sequences. Their role in angiotensin activity and nonhormone Netherlands), pp. 438-439. Downloaded by guest on September 29, 2021