Title: 120- and 160-kDa Receptors for Endogenous Mitogenic Peptide, Phytosulfokine-α, in Rice Plasma Membranes
Abstract: Plant cells in culture secrete a sulfated peptide named phytosulfokine-α (PSK-α), and this peptide induces the cell division and/or cell differentiation by means of specific high and low affinity receptors. Putative receptor proteins for this autocrine type growth factor were identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells. Incubation of membranes with a photoactivable 125I-labeled PSK-α analog, [N ε-(4-azidosalicyl)Lys5]PSK-α (AS-PSK-α), followed by UV irradiation resulted in specific labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis. The labeling of both bands was completely inhibited by unlabeled PSK-α and partially decreased by PSK-α analogs possessing moderate binding activities. In contrast, PSK-α analogs that have no biological activity showed no competition for125I-AS-PSK-α binding, confirming the specificity of binding proteins. Analysis of the affinity of 125I incorporation into the protein by ligand saturation experiments gave apparent K d values of 5.0 nm for the 120-kDa band and 5.4 nm for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site. Treatment of 125I-AS-PSK-α cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins contained approximately 10 kDa of N-linked oligosaccharides. Specific cross-linking of 125I-AS-PSK-α was also observed by using plasma membranes derived from carrot and tobacco cells, indicating the widespread occurrence of the binding proteins. Together, these data suggest that the 120- and 160-kDa proteins are PSK-α receptors that mediate the biological activities of PSK-α. Plant cells in culture secrete a sulfated peptide named phytosulfokine-α (PSK-α), and this peptide induces the cell division and/or cell differentiation by means of specific high and low affinity receptors. Putative receptor proteins for this autocrine type growth factor were identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells. Incubation of membranes with a photoactivable 125I-labeled PSK-α analog, [N ε-(4-azidosalicyl)Lys5]PSK-α (AS-PSK-α), followed by UV irradiation resulted in specific labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis. The labeling of both bands was completely inhibited by unlabeled PSK-α and partially decreased by PSK-α analogs possessing moderate binding activities. In contrast, PSK-α analogs that have no biological activity showed no competition for125I-AS-PSK-α binding, confirming the specificity of binding proteins. Analysis of the affinity of 125I incorporation into the protein by ligand saturation experiments gave apparent K d values of 5.0 nm for the 120-kDa band and 5.4 nm for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site. Treatment of 125I-AS-PSK-α cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins contained approximately 10 kDa of N-linked oligosaccharides. Specific cross-linking of 125I-AS-PSK-α was also observed by using plasma membranes derived from carrot and tobacco cells, indicating the widespread occurrence of the binding proteins. Together, these data suggest that the 120- and 160-kDa proteins are PSK-α receptors that mediate the biological activities of PSK-α. azidosalicylic peptide N-glycosidase F phytosulfokine phytosulfokine-α N-(9-fluorenyl)methoxycarbonyl high pressure liquid chromatography polyacrylamide gel electrophoresis N,N- dimethylformamide Plant cells in culture secrete a sulfated pentapeptide named phytosulfokine-α (PSK-α1; Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln) in response to externally added auxin and cytokinin, and PSK-α triggers cell proliferation at nanomolar concentrations in collaboration with plant hormones (1.Matsubayashi Y. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7623-7627Crossref PubMed Scopus (344) Google Scholar, 2.Matsubayashi Y. Morita A. Matsunaga E. Furuya A. Hanai N. Sakagami Y. Planta. 1999; 207: 559-565Crossref Scopus (54) Google Scholar). Therefore, dispersed cells cannot proliferate under low cell density conditions in which secreted PSK-α is diluted below the critical concentration with excess culture medium. This autocrine type peptide growth factor has been found in conditioned medium derived from both monocot and dicot cell cultures (3.Matsubayashi Y. Takagi L. Omura N. Morita A. Sakagami Y. Plant Physiol. (Bethesda). 1999; 120: 1043-1048Crossref PubMed Scopus (97) Google Scholar, 4.Hanai H. Matsuno T. Yamamoto M. Matsubayashi Y. Kobayashi T. Kamada H. Sakagami Y. Plant Cell Physiol. 2000; 41: 27-32Crossref PubMed Scopus (82) Google Scholar), implying that PSK-α is the general factor involved in plant cell growth. PSK-α also stimulates tracheary element differentiation of Zinniamesophyll cells (3.Matsubayashi Y. Takagi L. Omura N. Morita A. Sakagami Y. Plant Physiol. (Bethesda). 1999; 120: 1043-1048Crossref PubMed Scopus (97) Google Scholar) and somatic embryogenesis in carrots under defined conditions (5.Kobayashi T. Eun C.H. Hanai H. Matsubayashi Y. Sakagami Y. Kamada H. J. Exp. Bot. 1999; 50: 1123-1128Google Scholar).A cDNA encoding a PSK-α precursor has been isolated from cDNA library constructed using poly(A)+ mRNA purified from rice cells cultured for 10 days (6.Yang H. Matsubayashi Y. Nakamura K. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13560-13565Crossref PubMed Scopus (101) Google Scholar). The cDNA is 725 base pairs in length, and the 89-amino acid product, preprophytosulfokine, has a 22-amino acid hydrophobic region that resembles a cleavable leader peptide at its NH2 terminus. The PSK-α sequence occurs only once within the precursor, close to the COOH terminus. The critical importance of preprophytosulfokine in cell growth was shown by transforming rice cells with sense and antisense rice PSKgene that is regulated by the constitutive rice actin promoter. The sense transgenic cells divided about two times faster than the controls, whereas the antisense transgenic cells had decreased mitogenic activity.Evidence for the existence of high affinity binding sites for PSK-α in rice plasma membrane was provided initially by us using [35S]PSK-α (7.Matsubayashi Y. Takagi L. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13357-13362Crossref PubMed Scopus (114) Google Scholar) and later using [3H]PSK-α (8.Matsubayashi Y. Sakagami Y. Eur. J. Biochem. 1999; 262: 666-671Crossref PubMed Scopus (47) Google Scholar). The observed binding was saturable, reversible, and localized on the outer surface of the plasma membrane of rice cells. Because the specific binding was significantly altered by the external pH and ionic strength, the ligand-receptor binding may be mainly controlled by ionic interactions. Ligand saturation analysis using [3H]PSK-α revealed the existence of both high and low affinity binding sites with K d values of 1.4 and 27 nm, respectively. Specific binding activities for [3H]PSK-α have been detected in plasma membrane fractions derived from cell lines of many plant species containing carrot, maize, asparagus, and tomato, indicating the widespread occurrence of [3H]PSK-α-binding sites (8.Matsubayashi Y. Sakagami Y. Eur. J. Biochem. 1999; 262: 666-671Crossref PubMed Scopus (47) Google Scholar). At present, however, the molecular structures of PSK-α-binding sites remain rather unclear. To understand the molecular mechanism of how plant cells perceive and transduce the PSK-α signal, it is critically important to identify and characterize the receptor molecules that initially perceive the PSK-α signal.Derivatization of small peptide hormones with photoactivable groups has been utilized for characterization and purification of hormone receptors (9.Hazum E. Endocr. Rev. 1983; 4: 352-362Crossref PubMed Scopus (28) Google Scholar). In these cases, a key factor in the application of such functional groups may be how to modify peptides without loss of binding activity and biological activity. Structure-activity studies for PSK-α have shown that the active core of PSK-α is an NH2-terminal tripeptide containing two sulfated groups (10.Matsubayashi Y. Hanai H. Hara O. Sakagami Y. Biochem. Biophys. Res. Commun. 1996; 225: 209-214Crossref PubMed Scopus (62) Google Scholar). Thus, we focused on the COOH-terminal region of PSK-α for the functional derivatization of PSK-α.In this study, we prepared an 125I-labeled photoactivable PSK-α analog containing 4-azidosalicylic acid, [N ε-(4-azidosalicyl)Lys5]PSK-α, which possesses comparable binding activity with that of PSK-α in order to specifically label membrane proteins. After photoaffinity cross-linking, proteins of 120 and 160 kDa in rice plasma membranes were labeled with this ligand. These proteins fulfill the criteria expected for the high affinity PSK-α receptor(s) in terms of their affinity and specificity.DISCUSSIONAs part of a research program to characterize the PSK receptor protein, we prepared a fully active photoactivable probe that could be used in receptor visualization experiments through UV irradiation, SDS-PAGE, and autoradiography. In designing this analog, the ε amino group of [Lys5]PSK-α appeared particularly appropriate as the site of modification for the following reasons. First, structure-activity studies demonstrated that the COOH-terminal dipeptide of PSK-α does not play a critical role in the expression of its biological activity. Second, the allylazido moiety would not be expected to interfere with receptor binding due to its remote location relative to the message portion of PSK-α.The present study demonstrated cross-linking of125I-AS-PSK-α to rice plasma membrane proteins of 120 and 160 kDa. These proteins have all the properties expected of components of the PSK-α receptor that mediates proliferation and differentiation of plant cells. (a) The photoaffinity labeling of these two species occurs at biologically relevant concentrations of the ligand, in the range 0.1–10 nm; (b) the labeling of 120- and 160-kDa proteins is inhibited by native PSK-α in a dose-dependent manner; (c) the cross-linking is not affected by PSK-α analogs that have no biological activities; (d) the binding constants of 120- and 160-kDa proteins for125I-AS-PSK-α are at the nanomolar level, in agreement with the binding constant of [3H]PSK-α for the high affinity site in the rice plasma membrane fraction (1.4 nm) and also the ED50 of PSK-α (3.8 nm) determined by bioassay using asparagus mesophyll cells (1.Matsubayashi Y. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7623-7627Crossref PubMed Scopus (344) Google Scholar).Although we detected both high and low affinity binding sites in the rice plasma membrane fraction by the ligand binding assay using [3H]PSK-α (8.Matsubayashi Y. Sakagami Y. Eur. J. Biochem. 1999; 262: 666-671Crossref PubMed Scopus (47) Google Scholar), the photoaffinity labeling described here only revealed the presence of two binding proteins with high affinity binding constants. The absence of a low affinity protein detected by photoaffinity labeling might be explained by the following. Low affinity binding site could be produced by ligand-induced changes in receptor affinities and, therefore, not present at low ligand concentrations. A negatively cooperative model for hormone-receptor interaction has been reported for several mammal growth factor receptors (16.De Meyts P. Roth J. Neville Jr., D.M. Gavin III, J.R. Lesniak M.A. Biochem. Biophys. Res. Commun. 1973; 55: 154-161Crossref PubMed Scopus (470) Google Scholar, 17.Sutter A. Riopelle R.J. Harris-Warrick R.M. Shooter E.M. J. Biol. Chem. 1979; 254: 5972-5982Abstract Full Text PDF PubMed Google Scholar). We therefore tried to determine the binding constants of 125I-AS-PSK-α for the two proteins at high ligand concentrations, but unfortunately the high background precluded meaningful results. Alternatively, these may be two distinct PSK-α-binding sites that have different binding affinities in the rice plasma membrane fraction, but the low affinity site could not be labeled due to its conformational characteristics.The 160-kDa protein is not a multicomponent complex resulting from covalent cross-linking of the 120-kDa protein with other membrane proteins, since 125I-AS-PSK-α possesses only one photoactivable site which usually reacts with only one target molecule. This conclusion is further supported by the fact that the relative amount of labeling of the 120- and 160-kDa proteins did not change over a wide range of 125I-AS-PSK-α concentrations. The finding of two different PSK-α-binding proteins with different molecular weights can be interpreted in several ways. (a) Only the 160-kDa protein is the biologically relevant receptor, whereas the lower molecular mass 120-kDa protein is a proteolytic breakdown product. (b) The 120-kDa protein is a translation product of a truncated form of the mRNA encoding the 160-kDa protein. (c) The high affinity PSK-α receptor is associated with both of these two proteins without covalent links such as disulfide bonds. (d) Iodine radicals generated by UV irradiation may react at a site somewhat distant from the site of nitrene insertion (18.Van der Walt B. Cahnmann H.J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1492-1496Crossref PubMed Scopus (24) Google Scholar). (e) The two high affinity binding sites are structurally unrelated and are involved in different biological functions of PSK-α.In the case of mammal growth factor receptor, limited and specific proteolytic processes are known to transform the native insulin receptor (19.Massague J. Pilch P.F. Czech M.P. J. Biol. Chem. 1981; 256: 3182-3190Abstract Full Text PDF PubMed Google Scholar) and the epidermal growth factor (20.Cassel D. Glaser L. J. Biol. Chem. 1982; 257: 9845-9848Abstract Full Text PDF PubMed Google Scholar) to lower molecular weight forms. Moreover, it has been reported that in addition to the mature 175-kDa epidermal growth factor receptor, A431 cells also possess a 95-kDa form originating from such a truncated mRNA (21.Mayes E.L.V. Waterfield M.D. EMBO J. 1984; 3: 531-537Crossref PubMed Scopus (96) Google Scholar). In this context, interpretation a or b shown above may be a better explanation for the presence of two different PSK-α-binding proteins with different molecular weights.Several well characterized mammal growth factor receptors, including epidermal growth factor receptor (22.Soderquist A.M. Carpenter G. J. Biol. Chem. 1984; 259: 12586-12594Abstract Full Text PDF PubMed Google Scholar), platelet-derived growth factor receptor (23.Daniel T.O. Milfay D.F. Escobedo J. Williams L.T. J. Biol. Chem. 1987; 262: 9778-9784Abstract Full Text PDF PubMed Google Scholar), and insulin receptor (24.Reed B.C. Ronnett G.V. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2908-2912Crossref PubMed Scopus (64) Google Scholar), contain N-linked carbohydrate side chains. It has been demonstrated that core oligosaccharide addition is essential for the acquisition of epidermal growth factor binding activity (25.Slieker L.J. Lane M.D. J. Biol. Chem. 1985; 260: 687-690Abstract Full Text PDF PubMed Google Scholar). In addition, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin receptors (26.Ronnett G.V. Knutson V.P. Kohanski R.A. Simpson T.L. Lane M.D. J. Biol. Chem. 1984; 259: 4566-4575Abstract Full Text PDF PubMed Google Scholar). Although the function of the carbohydrate moiety in PSK-α receptors is still unclear, the presence of glycosylated side chain allows us to predict that immobilized lectins will be a useful tool in the purification of the PSK-α receptors.Occurrence of specifically 125I-AS-PSK-α cross-linked proteins with similar size across distantly related plant species, rice, carrot, and tobacco, is in good agreement with the widespread occurrence of PSK-α. Although the presence of a number of receptor-like membrane proteins involved in plant growth and development has been predicted based on sequence similarities (27.Clark S.E. Williams R.W. Meyerowitz E.M. Cell. 1997; 89: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar, 28.Hervé C. Dabos P. Galaud J.P. Rougé P. Lescure B. J. Mol. Biol. 1996; 258: 778-788Crossref PubMed Scopus (116) Google Scholar, 29.Li J. Chory J. Cell. 1997; 90: 929-938Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 30.Torii K.U. Mitsukawa N. Oosumi T. Matsuura Y. Yokohama R. Whittier R.F. Komeda Y. Plant Cell. 1996; 8: 735-746Crossref PubMed Scopus (589) Google Scholar) and biochemical characterization (31.Schaller G.E. Bleecker A.B. FEBS Lett. 1993; 333: 306-310Crossref PubMed Scopus (22) Google Scholar), little is known about the receptor ligand(s) that ultimately activates the receptor function through the receptor-ligand interaction. Recent evidence implies that plants, like animals, may actually make wide use of peptide signaling (1.Matsubayashi Y. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7623-7627Crossref PubMed Scopus (344) Google Scholar, 32.Marx J. Science. 1996; 273: 1338-1339Crossref PubMed Scopus (15) Google Scholar, 33.Scheer J.M. Ryan C.A. Plant Cell. 1999; 11: 1525-1536Crossref PubMed Scopus (116) Google Scholar), so that plant cell-to-cell communication is mediated by peptide-receptor interactions. Although further analysis of the PSK-α-binding proteins is needed to determine whether a relationship exists between the two proteins detected here, our work provides a basis for the purification and sequence analysis of PSK-α receptor(s) that perceive the extracellular peptide signal and transduce the intracellular secondary messengers activating sets of genes involved in plant cell proliferation and differentiation. Plant cells in culture secrete a sulfated pentapeptide named phytosulfokine-α (PSK-α1; Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln) in response to externally added auxin and cytokinin, and PSK-α triggers cell proliferation at nanomolar concentrations in collaboration with plant hormones (1.Matsubayashi Y. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7623-7627Crossref PubMed Scopus (344) Google Scholar, 2.Matsubayashi Y. Morita A. Matsunaga E. Furuya A. Hanai N. Sakagami Y. Planta. 1999; 207: 559-565Crossref Scopus (54) Google Scholar). Therefore, dispersed cells cannot proliferate under low cell density conditions in which secreted PSK-α is diluted below the critical concentration with excess culture medium. This autocrine type peptide growth factor has been found in conditioned medium derived from both monocot and dicot cell cultures (3.Matsubayashi Y. Takagi L. Omura N. Morita A. Sakagami Y. Plant Physiol. (Bethesda). 1999; 120: 1043-1048Crossref PubMed Scopus (97) Google Scholar, 4.Hanai H. Matsuno T. Yamamoto M. Matsubayashi Y. Kobayashi T. Kamada H. Sakagami Y. Plant Cell Physiol. 2000; 41: 27-32Crossref PubMed Scopus (82) Google Scholar), implying that PSK-α is the general factor involved in plant cell growth. PSK-α also stimulates tracheary element differentiation of Zinniamesophyll cells (3.Matsubayashi Y. Takagi L. Omura N. Morita A. Sakagami Y. Plant Physiol. (Bethesda). 1999; 120: 1043-1048Crossref PubMed Scopus (97) Google Scholar) and somatic embryogenesis in carrots under defined conditions (5.Kobayashi T. Eun C.H. Hanai H. Matsubayashi Y. Sakagami Y. Kamada H. J. Exp. Bot. 1999; 50: 1123-1128Google Scholar). A cDNA encoding a PSK-α precursor has been isolated from cDNA library constructed using poly(A)+ mRNA purified from rice cells cultured for 10 days (6.Yang H. Matsubayashi Y. Nakamura K. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13560-13565Crossref PubMed Scopus (101) Google Scholar). The cDNA is 725 base pairs in length, and the 89-amino acid product, preprophytosulfokine, has a 22-amino acid hydrophobic region that resembles a cleavable leader peptide at its NH2 terminus. The PSK-α sequence occurs only once within the precursor, close to the COOH terminus. The critical importance of preprophytosulfokine in cell growth was shown by transforming rice cells with sense and antisense rice PSKgene that is regulated by the constitutive rice actin promoter. The sense transgenic cells divided about two times faster than the controls, whereas the antisense transgenic cells had decreased mitogenic activity. Evidence for the existence of high affinity binding sites for PSK-α in rice plasma membrane was provided initially by us using [35S]PSK-α (7.Matsubayashi Y. Takagi L. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13357-13362Crossref PubMed Scopus (114) Google Scholar) and later using [3H]PSK-α (8.Matsubayashi Y. Sakagami Y. Eur. J. Biochem. 1999; 262: 666-671Crossref PubMed Scopus (47) Google Scholar). The observed binding was saturable, reversible, and localized on the outer surface of the plasma membrane of rice cells. Because the specific binding was significantly altered by the external pH and ionic strength, the ligand-receptor binding may be mainly controlled by ionic interactions. Ligand saturation analysis using [3H]PSK-α revealed the existence of both high and low affinity binding sites with K d values of 1.4 and 27 nm, respectively. Specific binding activities for [3H]PSK-α have been detected in plasma membrane fractions derived from cell lines of many plant species containing carrot, maize, asparagus, and tomato, indicating the widespread occurrence of [3H]PSK-α-binding sites (8.Matsubayashi Y. Sakagami Y. Eur. J. Biochem. 1999; 262: 666-671Crossref PubMed Scopus (47) Google Scholar). At present, however, the molecular structures of PSK-α-binding sites remain rather unclear. To understand the molecular mechanism of how plant cells perceive and transduce the PSK-α signal, it is critically important to identify and characterize the receptor molecules that initially perceive the PSK-α signal. Derivatization of small peptide hormones with photoactivable groups has been utilized for characterization and purification of hormone receptors (9.Hazum E. Endocr. Rev. 1983; 4: 352-362Crossref PubMed Scopus (28) Google Scholar). In these cases, a key factor in the application of such functional groups may be how to modify peptides without loss of binding activity and biological activity. Structure-activity studies for PSK-α have shown that the active core of PSK-α is an NH2-terminal tripeptide containing two sulfated groups (10.Matsubayashi Y. Hanai H. Hara O. Sakagami Y. Biochem. Biophys. Res. Commun. 1996; 225: 209-214Crossref PubMed Scopus (62) Google Scholar). Thus, we focused on the COOH-terminal region of PSK-α for the functional derivatization of PSK-α. In this study, we prepared an 125I-labeled photoactivable PSK-α analog containing 4-azidosalicylic acid, [N ε-(4-azidosalicyl)Lys5]PSK-α, which possesses comparable binding activity with that of PSK-α in order to specifically label membrane proteins. After photoaffinity cross-linking, proteins of 120 and 160 kDa in rice plasma membranes were labeled with this ligand. These proteins fulfill the criteria expected for the high affinity PSK-α receptor(s) in terms of their affinity and specificity. DISCUSSIONAs part of a research program to characterize the PSK receptor protein, we prepared a fully active photoactivable probe that could be used in receptor visualization experiments through UV irradiation, SDS-PAGE, and autoradiography. In designing this analog, the ε amino group of [Lys5]PSK-α appeared particularly appropriate as the site of modification for the following reasons. First, structure-activity studies demonstrated that the COOH-terminal dipeptide of PSK-α does not play a critical role in the expression of its biological activity. Second, the allylazido moiety would not be expected to interfere with receptor binding due to its remote location relative to the message portion of PSK-α.The present study demonstrated cross-linking of125I-AS-PSK-α to rice plasma membrane proteins of 120 and 160 kDa. These proteins have all the properties expected of components of the PSK-α receptor that mediates proliferation and differentiation of plant cells. (a) The photoaffinity labeling of these two species occurs at biologically relevant concentrations of the ligand, in the range 0.1–10 nm; (b) the labeling of 120- and 160-kDa proteins is inhibited by native PSK-α in a dose-dependent manner; (c) the cross-linking is not affected by PSK-α analogs that have no biological activities; (d) the binding constants of 120- and 160-kDa proteins for125I-AS-PSK-α are at the nanomolar level, in agreement with the binding constant of [3H]PSK-α for the high affinity site in the rice plasma membrane fraction (1.4 nm) and also the ED50 of PSK-α (3.8 nm) determined by bioassay using asparagus mesophyll cells (1.Matsubayashi Y. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7623-7627Crossref PubMed Scopus (344) Google Scholar).Although we detected both high and low affinity binding sites in the rice plasma membrane fraction by the ligand binding assay using [3H]PSK-α (8.Matsubayashi Y. Sakagami Y. Eur. J. Biochem. 1999; 262: 666-671Crossref PubMed Scopus (47) Google Scholar), the photoaffinity labeling described here only revealed the presence of two binding proteins with high affinity binding constants. The absence of a low affinity protein detected by photoaffinity labeling might be explained by the following. Low affinity binding site could be produced by ligand-induced changes in receptor affinities and, therefore, not present at low ligand concentrations. A negatively cooperative model for hormone-receptor interaction has been reported for several mammal growth factor receptors (16.De Meyts P. Roth J. Neville Jr., D.M. Gavin III, J.R. Lesniak M.A. Biochem. Biophys. Res. Commun. 1973; 55: 154-161Crossref PubMed Scopus (470) Google Scholar, 17.Sutter A. Riopelle R.J. Harris-Warrick R.M. Shooter E.M. J. Biol. Chem. 1979; 254: 5972-5982Abstract Full Text PDF PubMed Google Scholar). We therefore tried to determine the binding constants of 125I-AS-PSK-α for the two proteins at high ligand concentrations, but unfortunately the high background precluded meaningful results. Alternatively, these may be two distinct PSK-α-binding sites that have different binding affinities in the rice plasma membrane fraction, but the low affinity site could not be labeled due to its conformational characteristics.The 160-kDa protein is not a multicomponent complex resulting from covalent cross-linking of the 120-kDa protein with other membrane proteins, since 125I-AS-PSK-α possesses only one photoactivable site which usually reacts with only one target molecule. This conclusion is further supported by the fact that the relative amount of labeling of the 120- and 160-kDa proteins did not change over a wide range of 125I-AS-PSK-α concentrations. The finding of two different PSK-α-binding proteins with different molecular weights can be interpreted in several ways. (a) Only the 160-kDa protein is the biologically relevant receptor, whereas the lower molecular mass 120-kDa protein is a proteolytic breakdown product. (b) The 120-kDa protein is a translation product of a truncated form of the mRNA encoding the 160-kDa protein. (c) The high affinity PSK-α receptor is associated with both of these two proteins without covalent links such as disulfide bonds. (d) Iodine radicals generated by UV irradiation may react at a site somewhat distant from the site of nitrene insertion (18.Van der Walt B. Cahnmann H.J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1492-1496Crossref PubMed Scopus (24) Google Scholar). (e) The two high affinity binding sites are structurally unrelated and are involved in different biological functions of PSK-α.In the case of mammal growth factor receptor, limited and specific proteolytic processes are known to transform the native insulin receptor (19.Massague J. Pilch P.F. Czech M.P. J. Biol. Chem. 1981; 256: 3182-3190Abstract Full Text PDF PubMed Google Scholar) and the epidermal growth factor (20.Cassel D. Glaser L. J. Biol. Chem. 1982; 257: 9845-9848Abstract Full Text PDF PubMed Google Scholar) to lower molecular weight forms. Moreover, it has been reported that in addition to the mature 175-kDa epidermal growth factor receptor, A431 cells also possess a 95-kDa form originating from such a truncated mRNA (21.Mayes E.L.V. Waterfield M.D. EMBO J. 1984; 3: 531-537Crossref PubMed Scopus (96) Google Scholar). In this context, interpretation a or b shown above may be a better explanation for the presence of two different PSK-α-binding proteins with different molecular weights.Several well characterized mammal growth factor receptors, including epidermal growth factor receptor (22.Soderquist A.M. Carpenter G. J. Biol. Chem. 1984; 259: 12586-12594Abstract Full Text PDF PubMed Google Scholar), platelet-derived growth factor receptor (23.Daniel T.O. Milfay D.F. Escobedo J. Williams L.T. J. Biol. Chem. 1987; 262: 9778-9784Abstract Full Text PDF PubMed Google Scholar), and insulin receptor (24.Reed B.C. Ronnett G.V. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2908-2912Crossref PubMed Scopus (64) Google Scholar), contain N-linked carbohydrate side chains. It has been demonstrated that core oligosaccharide addition is essential for the acquisition of epidermal growth factor binding activity (25.Slieker L.J. Lane M.D. J. Biol. Chem. 1985; 260: 687-690Abstract Full Text PDF PubMed Google Scholar). In addition, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin receptors (26.Ronnett G.V. Knutson V.P. Kohanski R.A. Simpson T.L. Lane M.D. J. Biol. Chem. 1984; 259: 4566-4575Abstract Full Text PDF PubMed Google Scholar). Although the function of the carbohydrate moiety in PSK-α receptors is still unclear, the presence of glycosylated side chain allows us to predict that immobilized lectins will be a useful tool in the purification of the PSK-α receptors.Occurrence of specifically 125I-AS-PSK-α cross-linked proteins with similar size across distantly related plant species, rice, carrot, and tobacco, is in good agreement with the widespread occurrence of PSK-α. Although the presence of a number of receptor-like membrane proteins involved in plant growth and development has been predicted based on sequence similarities (27.Clark S.E. Williams R.W. Meyerowitz E.M. Cell. 1997; 89: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar, 28.Hervé C. Dabos P. Galaud J.P. Rougé P. Lescure B. J. Mol. Biol. 1996; 258: 778-788Crossref PubMed Scopus (116) Google Scholar, 29.Li J. Chory J. Cell. 1997; 90: 929-938Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 30.Torii K.U. Mitsukawa N. Oosumi T. Matsuura Y. Yokohama R. Whittier R.F. Komeda Y. Plant Cell. 1996; 8: 735-746Crossref PubMed Scopus (589) Google Scholar) and biochemical characterization (31.Schaller G.E. Bleecker A.B. FEBS Lett. 1993; 333: 306-310Crossref PubMed Scopus (22) Google Scholar), little is known about the receptor ligand(s) that ultimately activates the receptor function through the receptor-ligand interaction. Recent evidence implies that plants, like animals, may actually make wide use of peptide signaling (1.Matsubayashi Y. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7623-7627Crossref PubMed Scopus (344) Google Scholar, 32.Marx J. Science. 1996; 273: 1338-1339Crossref PubMed Scopus (15) Google Scholar, 33.Scheer J.M. Ryan C.A. Plant Cell. 1999; 11: 1525-1536Crossref PubMed Scopus (116) Google Scholar), so that plant cell-to-cell communication is mediated by peptide-receptor interactions. Although further analysis of the PSK-α-binding proteins is needed to determine whether a relationship exists between the two proteins detected here, our work provides a basis for the purification and sequence analysis of PSK-α receptor(s) that perceive the extracellular peptide signal and transduce the intracellular secondary messengers activating sets of genes involved in plant cell proliferation and differentiation. As part of a research program to characterize the PSK receptor protein, we prepared a fully active photoactivable probe that could be used in receptor visualization experiments through UV irradiation, SDS-PAGE, and autoradiography. In designing this analog, the ε amino group of [Lys5]PSK-α appeared particularly appropriate as the site of modification for the following reasons. First, structure-activity studies demonstrated that the COOH-terminal dipeptide of PSK-α does not play a critical role in the expression of its biological activity. Second, the allylazido moiety would not be expected to interfere with receptor binding due to its remote location relative to the message portion of PSK-α. The present study demonstrated cross-linking of125I-AS-PSK-α to rice plasma membrane proteins of 120 and 160 kDa. These proteins have all the properties expected of components of the PSK-α receptor that mediates proliferation and differentiation of plant cells. (a) The photoaffinity labeling of these two species occurs at biologically relevant concentrations of the ligand, in the range 0.1–10 nm; (b) the labeling of 120- and 160-kDa proteins is inhibited by native PSK-α in a dose-dependent manner; (c) the cross-linking is not affected by PSK-α analogs that have no biological activities; (d) the binding constants of 120- and 160-kDa proteins for125I-AS-PSK-α are at the nanomolar level, in agreement with the binding constant of [3H]PSK-α for the high affinity site in the rice plasma membrane fraction (1.4 nm) and also the ED50 of PSK-α (3.8 nm) determined by bioassay using asparagus mesophyll cells (1.Matsubayashi Y. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7623-7627Crossref PubMed Scopus (344) Google Scholar). Although we detected both high and low affinity binding sites in the rice plasma membrane fraction by the ligand binding assay using [3H]PSK-α (8.Matsubayashi Y. Sakagami Y. Eur. J. Biochem. 1999; 262: 666-671Crossref PubMed Scopus (47) Google Scholar), the photoaffinity labeling described here only revealed the presence of two binding proteins with high affinity binding constants. The absence of a low affinity protein detected by photoaffinity labeling might be explained by the following. Low affinity binding site could be produced by ligand-induced changes in receptor affinities and, therefore, not present at low ligand concentrations. A negatively cooperative model for hormone-receptor interaction has been reported for several mammal growth factor receptors (16.De Meyts P. Roth J. Neville Jr., D.M. Gavin III, J.R. Lesniak M.A. Biochem. Biophys. Res. Commun. 1973; 55: 154-161Crossref PubMed Scopus (470) Google Scholar, 17.Sutter A. Riopelle R.J. Harris-Warrick R.M. Shooter E.M. J. Biol. Chem. 1979; 254: 5972-5982Abstract Full Text PDF PubMed Google Scholar). We therefore tried to determine the binding constants of 125I-AS-PSK-α for the two proteins at high ligand concentrations, but unfortunately the high background precluded meaningful results. Alternatively, these may be two distinct PSK-α-binding sites that have different binding affinities in the rice plasma membrane fraction, but the low affinity site could not be labeled due to its conformational characteristics. The 160-kDa protein is not a multicomponent complex resulting from covalent cross-linking of the 120-kDa protein with other membrane proteins, since 125I-AS-PSK-α possesses only one photoactivable site which usually reacts with only one target molecule. This conclusion is further supported by the fact that the relative amount of labeling of the 120- and 160-kDa proteins did not change over a wide range of 125I-AS-PSK-α concentrations. The finding of two different PSK-α-binding proteins with different molecular weights can be interpreted in several ways. (a) Only the 160-kDa protein is the biologically relevant receptor, whereas the lower molecular mass 120-kDa protein is a proteolytic breakdown product. (b) The 120-kDa protein is a translation product of a truncated form of the mRNA encoding the 160-kDa protein. (c) The high affinity PSK-α receptor is associated with both of these two proteins without covalent links such as disulfide bonds. (d) Iodine radicals generated by UV irradiation may react at a site somewhat distant from the site of nitrene insertion (18.Van der Walt B. Cahnmann H.J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1492-1496Crossref PubMed Scopus (24) Google Scholar). (e) The two high affinity binding sites are structurally unrelated and are involved in different biological functions of PSK-α. In the case of mammal growth factor receptor, limited and specific proteolytic processes are known to transform the native insulin receptor (19.Massague J. Pilch P.F. Czech M.P. J. Biol. Chem. 1981; 256: 3182-3190Abstract Full Text PDF PubMed Google Scholar) and the epidermal growth factor (20.Cassel D. Glaser L. J. Biol. Chem. 1982; 257: 9845-9848Abstract Full Text PDF PubMed Google Scholar) to lower molecular weight forms. Moreover, it has been reported that in addition to the mature 175-kDa epidermal growth factor receptor, A431 cells also possess a 95-kDa form originating from such a truncated mRNA (21.Mayes E.L.V. Waterfield M.D. EMBO J. 1984; 3: 531-537Crossref PubMed Scopus (96) Google Scholar). In this context, interpretation a or b shown above may be a better explanation for the presence of two different PSK-α-binding proteins with different molecular weights. Several well characterized mammal growth factor receptors, including epidermal growth factor receptor (22.Soderquist A.M. Carpenter G. J. Biol. Chem. 1984; 259: 12586-12594Abstract Full Text PDF PubMed Google Scholar), platelet-derived growth factor receptor (23.Daniel T.O. Milfay D.F. Escobedo J. Williams L.T. J. Biol. Chem. 1987; 262: 9778-9784Abstract Full Text PDF PubMed Google Scholar), and insulin receptor (24.Reed B.C. Ronnett G.V. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2908-2912Crossref PubMed Scopus (64) Google Scholar), contain N-linked carbohydrate side chains. It has been demonstrated that core oligosaccharide addition is essential for the acquisition of epidermal growth factor binding activity (25.Slieker L.J. Lane M.D. J. Biol. Chem. 1985; 260: 687-690Abstract Full Text PDF PubMed Google Scholar). In addition, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin receptors (26.Ronnett G.V. Knutson V.P. Kohanski R.A. Simpson T.L. Lane M.D. J. Biol. Chem. 1984; 259: 4566-4575Abstract Full Text PDF PubMed Google Scholar). Although the function of the carbohydrate moiety in PSK-α receptors is still unclear, the presence of glycosylated side chain allows us to predict that immobilized lectins will be a useful tool in the purification of the PSK-α receptors. Occurrence of specifically 125I-AS-PSK-α cross-linked proteins with similar size across distantly related plant species, rice, carrot, and tobacco, is in good agreement with the widespread occurrence of PSK-α. Although the presence of a number of receptor-like membrane proteins involved in plant growth and development has been predicted based on sequence similarities (27.Clark S.E. Williams R.W. Meyerowitz E.M. Cell. 1997; 89: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar, 28.Hervé C. Dabos P. Galaud J.P. Rougé P. Lescure B. J. Mol. Biol. 1996; 258: 778-788Crossref PubMed Scopus (116) Google Scholar, 29.Li J. Chory J. Cell. 1997; 90: 929-938Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 30.Torii K.U. Mitsukawa N. Oosumi T. Matsuura Y. Yokohama R. Whittier R.F. Komeda Y. Plant Cell. 1996; 8: 735-746Crossref PubMed Scopus (589) Google Scholar) and biochemical characterization (31.Schaller G.E. Bleecker A.B. FEBS Lett. 1993; 333: 306-310Crossref PubMed Scopus (22) Google Scholar), little is known about the receptor ligand(s) that ultimately activates the receptor function through the receptor-ligand interaction. Recent evidence implies that plants, like animals, may actually make wide use of peptide signaling (1.Matsubayashi Y. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7623-7627Crossref PubMed Scopus (344) Google Scholar, 32.Marx J. Science. 1996; 273: 1338-1339Crossref PubMed Scopus (15) Google Scholar, 33.Scheer J.M. Ryan C.A. Plant Cell. 1999; 11: 1525-1536Crossref PubMed Scopus (116) Google Scholar), so that plant cell-to-cell communication is mediated by peptide-receptor interactions. Although further analysis of the PSK-α-binding proteins is needed to determine whether a relationship exists between the two proteins detected here, our work provides a basis for the purification and sequence analysis of PSK-α receptor(s) that perceive the extracellular peptide signal and transduce the intracellular secondary messengers activating sets of genes involved in plant cell proliferation and differentiation. We thank Dr. K. Syono (Japan Women's University) for providing rice (Oc) suspension cell, Dr. H. Kamada (University of Tsukuba) for carrot (NC), and Dr. K. Nakamura (Nagoya University) for tobacco (BY-2).