- Email: [email protected]
The subunit structure of the insulin receptor and molecular interactions with major histocompatibility complex antigens
BIOCttlMIE, 1985, 67, 1155-1159
The subunit structure of the insulin receptor and molecular interactions with major histocompatibility complex antigens. Max F E H L M A N N , Yolande CHVATCHKO, Dietrich B R A N D E N B U R G * , Emmanuel V A N O B B E R G H E N and Nicole BROSSETrE.
INSERM. U145, Facultd de M(dechle (Pasteur), 06034, Nice Cedex. * Deutsches Wollforschungsinstitut, Aachen, RFA. (Refu le 22-5-1985, accept~ apr~s r(vision le 21-6-1985).
R6sum6 - - Le rdcepteur hlsulinique a dtd marqud h l'aide d'un ddrivd photor(actif de l'insuline (marquage spdcifique des sous-unitds ct) et par autophosphorylation en prdsence d'insuline (marquage spdcifique des sous-unitds fl). Les rdsultats montrent que le rdcepteur de l'insuline existe sous diffdrentes formes oligomdriques des sous-unitds ~t et fl lides entre elles par des ponts disulfure. De plus, le rdcepteur hTsulinique est dtroitement associd attx antigdnes de classe I du complexe majeur d'histocompatibilitd. Mots-cl~s : marquage par photoaffinit6 / tyrosine kinase / immnnopr~eipitation.
S u m m a r y - - Insulin receptors were labeled with 1251-photoreactive hlsulin (specifically labeling ct-subunits) and by insulin-sthnulated atttophosphorylation (specifically labeling fl-subunits). The results show that the hlsulin receptor exists under different free and disulfide-linked combinations of ct and fl subunits. Moreover, the insulin receptor is closely associated to class I antigens of the major histocompatibility complex to form a high molecular weight multi-molecular membrane complex. Key-words : photoaffinity labeling / tyrosine-kinase / immunoprecipitation.
Insulin, synthesized and secreted by pancreatic I~ cells, is involved at the level of its target cells in the regulation of a considerable number of metabolic and growth events. The first limiting step in the cellular responses to insulin is the recognition of the hormone by specific receptors expressed at the surface of the target cells. This binding step is followed by a large array o f biological effects taking place both at the plasma membrane level and at intracellular sites. The hormone-receptor interaction occurring at the cell surface has been characterized in detail [1, 2], but the mechanism of the hormonal signal transmis-
sion from the membrane to intracellular sites has not as yet been elucidated. Results accumulated during the last five years suggest that the clue to this problem resides in the structure of the insulin receptor itself. Indeed, antibodies specifically recognizing the insulin receptor are able to mimick most of the insulin biological effects, thus indicating that the information for hormone signalling is contained in the t-eceptor molecule and not in the ligand [3]. The detailed knowledge o f the structure of the insulin receptor thus seems to constitute a prerequisite to unravel the mechanism of insulin action.
1156
AL Fehlmann and coll.
Oiigomeric structure of the insulin receptor In most tissues and species investigated so far the insulin receptor is composed of at least 2 types of subunits linked together by disulfide bridges. The ct-subunit (Mr 130,000), which contains the insulin binding site, can be labeled by photoreactive insulin analogues [4, 5, 6] or with bifunctional crosslinking agents [7]. The [3-subunit (Mr 95,000) contains a tyrosine-kinase activity capable of autophosphorylation reactions in the presence of insulin [8, 9]. Thus by using these two biological properties of the insulin receptor, it is possible to selectively label the ct-subunits (by affinity labeling) and the [3-subunits (by autophosphorylation). When rat hepatoma cells (Fao) were incubated with ~'-5I-photoreactive insulin, only one protein (Mr 130,000) was labeled, as
shown in Figure 1 (lane C). This labeling was specific since it was inhibited by an excess of unlabeled insulin (Fig. 1, lane D). This protein corresponds to the ct-subunit of the hepatic insulin receptor containing the hormone binding site. When insulin receptors, partially purified by affinity chromatography, were incubated with (y-3'P)ATP and insulin, one major labeled protein (Mr 95,000) was observed (Fig. 1, lane B). This protein, which is not labeled in the absence .of insulin (Fig. 1, lane A) corresponds to the 13-subunit of the insulin receptor. When the labeling patterns obtained with mI-photoreactive insulin (ct-subunits) and (y-3Zp)ATP (fl-subunits) were compared after polyacrylamide gel electrophoresis under nonreducing conditions, the insulin receptor was found under different oligomeric forms (Fig. 2). These forms correspond to different combinations
32p 32 p e R ~
OR-- V
"° .
200--
94--
MrX
"'°'213
m
Ib,
116--
125 i
1251
0(2""
o( ----- I
." ~''
m
O--p
oo-
67~.
116--
43 -¸
94--
302014_=-- ..... 1() 3 A B
"" I O
O
67-m
dlBL C
D
I
M r × l O -3
A
B
C
D
FIG. I. -- Labeling of insufin receptors by autophosphoo'lation (J:P) amt with a photoreactive insufin analogue (~:~I).
FIG. 2. -- Labeling of insulin receptors by atttophosphorvlation (3:p) and with a photoreactive insulin analogue (t:~l)."
Insulin receptors were partially purified by affinity chromatography and phosphorylated with (yJ"P)ATP in the absence (A) or presence of 10-TM insulin (B). For photoaffinity labeling, cells were incubated with ':51-photoreactive insulin in the absence (C) or presence of 10-6M unlabeled insulin (D), UV-irradiated and directly solubilized in boiling SDS. Labeled proteins were analyzed by SDS-PAGE under reducing conditions using 7,5 % acrylamide gels, followed by autoradiography.
Insulin receptors were purified by lectin affinity chromatography and exposed to a control serum (A) or to a serum containing autoantibodies to the insulin receptor (B). After addition of protein A the immunoprecipitates were phosphorylated with (¥J:P)ATP. For photoaffinity labeling, cells were incubated with IZ~l-photoreactive insulin in the absence (t~) or presence of 10-6M unlabeled insulin (D), UV-irradiated and directly solubilized in boiling SDS. Labeled proteins were analyzed by SDS-PAGE under non-reducing conditions using 5 % acrylamide gels, followed by autoradiography.
Structure of insulin receptors of a- and 13-subunits : free ct, free 13, a13, a2, a213, and ct2 132 with apparent molecular masses of 95, 130, 220, 250, 350 and 450 kDa, respectively [10]. By contrast the I]2 combination was never found. It can thus be concluded that the insulin receptor is an immunoglobin-like heterotetramer, with 2 "heavy" chains (ct) linked together by disulfide bridges, each "heavy" chain being itself linked to a "light" chain (13) also by a S-S bridge (see Fig. 5). The presence of free insulin receptor subunits in native membranes of insulin-responsive cells has already been reported [11] but their contribution in receptor function and receptor regulation is not as yet totally understood. It seems, however, that oligomeric forms are preferentially subjected to receptor down-regulation when cells are exposed to high concentrations of insulin and that t h e number of free subunits is directly correlated to the average affinity of the receptor [11]. More complex models of the insulin receptor have been proposed on the basis of data obtained
i157
with photoreactive insulin analogues [12, 13]. Thus, the photoreactive analogue B29-Napa-insulin (the photoreactive residue being attached in position 29 of the insulin B chain) labels the ct-subunit (130 kDa) of the insulin receptor and in addition 4 proteins with molecular masses of 95, 85, 55 and 45 kDa (Fig. 3, lane C). The labeling of these proteins is specific (inhibited by unlabeled insulin : Fig. 3, lane D) and moreover, these proteins can be precipitated by anti-receptor antibodies. By contrast, the analogue B2-Napa-des PheBl-insulin labels only the u-subunit and a protein at 95 kDa (Fig. 3, lanes A and
B). It is therefore possible that in plasma membranes the insulin receptor is associated to other membrane proteins that can participate to the binding of insulin, since residue B29 is located in the insulin domain involved in the binding to the receptor.
Interactions with antigens of the major histocompatibility complex ( M H C ) A
B
C
D
E ....
OR-
F
200--
116--
-m,
9
94--
g
4
¢ 13o
67I
43--
tQ
tB
II
B1
'
II
B29
J
B2
FIG. 3. -- Labeling o f insulin receptors by different photoreactive analogues. Liver plasma membranes from C3H mice were incubated with BI-Napa-insulin (BI), B29-Napa-insulin (B29) and B2-Napa-des-Phe -8~ insulin (B2) (Napa : 2-nitro, 4-azidopbenylacetyl) in the absence (A, C, E) or in the presence (B, D, F) of 10 -6 unlabeled insulin, then UV-irradiated. Proteins were solubilized and analyzed as indicated in the legend to Figure 1.
Identification of membrane proteins associated with the insulin receptor can be undertaken by screening the effect of various anti-membrane protein antibodies on the binding of insulin to its receptor. Using this approach it has been demonstrated that insulin binding to HL60 cells (human promyelocytic cells) is inhibited by anti-HLA antibodies [14]. Figure 4 shows that insulin receptors from mouse liver plasma membranes, labeled with ~25I-photoreactive insulin, are precipitated by monoclonal antibodies reacting with H-2K (a heavy chain of class I antigens of the mouse MHC). Approximately 15% of the receptors which are precipitated by anti-receptor antibodies (Fig. 4, lane A) are precipitated by different anti H-2 antibodies (Fig. 4, lanes B-D), whereas no receptors are precipitated by an equivalent amount of normal mouse IgG's (Fig. 4, lane E) nor by a control serum (Fig. 4, lane F). This precipitation of insulin receptors by anti-H2 antibodies can be due to the presence of common epitopes on insulin receptors and H2 antigens (cross-reactivity) or to the formation of a molecular complex between those 2 proteins (coprecipitation). The experiment shown in Figure 4 indicates that 2 different monocional antibodies (la-
M. Fehlmann and coll.
1158 A
B
C
D
em
m
E
F
0R20011694-
O
/
¢ 13C
=,,
67--
43-ATP
30Mrxl() 3 ;
/
/
m- v 'as'
FIG. 4. -- hnnntnoprecipitation of insulin receptors by attti-H2 antibodies. Liver plasma membranes from CH3 (H-2~) mice were labeled with photoreactive insulin, solubilized in 1% Nonidet P-40 and incubated with : (A) serum containing anti-insulin receptor antibodies; (B) monoclonal antibodies reacting with Kk, D~ (H-100-30/23); (C) and (D) monoclonal antibodies reacting with Kk (H-100-5/28 and 11-4-1, respectively); (E) normal mouse IgG's; and (F) control serum. After addition of protein A, immunoprecipitates ~vereanalyzed by gel electrophoresis as indicated in the legend to Figure I.
nes B and C), known to react with distinct antigenic determinants [15, 16], are both capable of precipitating the receptor, which favors the coprecipitation possibility. Molecular association has been demonstrated by sequential immunoprecipitations (Fehlmann et al. submitted), and by using different mouse strains [17, 18]. These results thus show that in the hepatocyte plasma membrane the insulin receptor is closely linked to MHC antigens (Fig. 5). In the immune system MHC antigens are recognized by T lymphocytes in association with other membrane antigens. This process of corecognition is involved in the lysis of virus-infected cells by cytotoxic T cells (for class I MHC antigens) and in the stimulation of helper T cells by antigen presenting cells (for class II MHC antigens). The initial molecular event in this co-recognition process seems to reside in a physical interaction between MHC proteins and foreign antigens. Similarly, molecular interactions are believed to be involved in different non-
ADP
FIG. 5. -- The insulin receptor is associated to Class 1 (H-2K) antigens of the major histocompatibilio" complex in mouse liter membranes. immunological functions of M H C antigens including cell adhesion [20] and hormone action [21,22,23]. Anti-HLA antibodies have been shown to inhibit E G F [22] and insulin [14] binding to human cells in culture. Correspondingly, anti-fl2 microglobulin (the light chain o f class I MHC antigens) inhibits platelet aggregation induced by thrombin [23]. These results, together with the demonstration of a physical interaction between insulin receptors and class I M H C antigens, suggest that MHC antigens are directly involved in the transmission o f the hormonal signals triggered by the binding of hormones to their specific receptors. The key question is now the possible relationship between the kinase activity associated with the insulin receptor and MHC antigens. HLA antigens have been shown to be phosphorylated by the tyrosine-kinase associated with the Rous sarcoma virus [24] and similarly, preliminary results in our laboratory have shown that H-2 antigens are phosphorylated by the insulin receptor tyrosine-kinase. These observations may enable to reevaluate the biological functions o f the MHC, especially in the etiology of diabetes and in the immune tolerance of tumors that are under the control of growth and transformation factors.
Acknowledgments We are indebted to Anne-Marie Schmitt-Verhulst, Nicole Kiger, David Sachs and Ronald Kahn for helpful discussions and for the gift o f the different antibodies that have been used in this study.
Structure o f insulin receptors
I 159
REFERENCES 1. Kahn, C.R. (1976) J. Cell BioL. 70, 261-286. 2. Roth, J. (1979) In : Endocrinology (De Groot, ed.) Vol. 3, pp. 2037-2054, Grune and Stratton, New-York. 3. Kahn, C.R., Baird, K.L., Flier, J.S., Grunfeld, C., Harmon, J.T., Harrison, L.C., Karlsson, F.A., Kasuga, M., King, G., Lang, U.C., Podskalny, J.M. & Van Obberghen, E. (1981) Rec. Prog. Horm. Res., 37, 477-538. 4. Yip, C.C., Yeung, C.W.T. & Moule, M.L. (1980) Biochemistr)" 19, 70-76. 5. Wisher, M.H., Baron, M.D., Jones, R.H., S6nksen, P.H., Saunders, D.J., Thamm, P. & Brandenburg, D. (1980) Biochem. Biophys. Res. Commun., 92, 492-498. 6. Fehlmann, M., Le Marchand-Brustel, Y., Van Obberghen, E., Brandenburg, D. & Freychet, P. (1982) Diabetologia. 23, 440-444. 7. Pilch, P.F. & Czech, M.P. (1980) J. Biol. Chem., 25, 1722-1731. 8. Kasuga, M., Karlsson, F.A. & Kahn, C.R. (1982) Science, 215, 185-187. 9. Van Obberghen, E., Rossi, B., Kowalski, A., Gazzano, H. & Ponzio, G. (1983), Proe. Natl. Acad. Sci. USA, 80, 945-949. 10. Chvatchko, Y., Gazzano, H., Van Obberghen, E. & Fehlmann, M. (1984), Mol. Cell. Endocrinol., 36, 59-65.
il. Crettaz, M., Jialal, I., Kasuga, M. & Kahn, C.R. (1984) .L Biol. Chem., 259, 11543-11549. 12. Baron, M.D. & S6nksen, P.H. (1983) Biochem. J., 212, 79-84. 13. Yip, C.C. & Moule, M.L. (1983) Diabetes, 32, 760-767. 14. Simonsen, M. & Olsson, L. (1983) Ann. Immunol. (Paris), 134, 85-92. 15. Lemke, H., H~immerling, G.J. & H~mmerling, U. (1970) 1mmunol. Rev., 47, 175-182. 16. Oi, V.T., Jones, P.P., Goding, J.W. & Herzenberg, L.A. (1978) Curr. Top. MicrobioL Immunol., 81, 115-121. 17. Chvatchko, Y., Van Obberghen, E., Kiger, N. & Fehlmann M. (1983) FEBS Left., 163, 207-211. 18. Brossette, N., Van Obberghen, E. & Fehlmann, M. (1984) Diabetologia, 27, 74-76. 19. Edidin, M. (1983) Immunology Today 4, 269-270. 20. Curtis, A.S. & Rooney, P. (1979) Nature, 281, 222-223. 21. Lafuse, W. & Edidin, M. (1980) Biochemistry, 19, 49-54. 22. Schreiber, A.B., Schlessinger, J. & Edidin, M. (1984) J. Cell Biol., 98, 725-731. 23. Curry, R.A., Messner, R.P., Johnson, G.J. (1984) Science, 224, 509-511. 24. Guild, B., Erikson, R.L. & Strominger, J.L. (1983) Proc. NatL Acad. ScL USA, 80, 2894-2898.
The subunit structure of the insulin receptor and molecular interactions with major histocompatibility complex antigens. Max F E H L M A N N , Yolande CHVATCHKO, Dietrich B R A N D E N B U R G * , Emmanuel V A N O B B E R G H E N and Nicole BROSSETrE.
INSERM. U145, Facultd de M(dechle (Pasteur), 06034, Nice Cedex. * Deutsches Wollforschungsinstitut, Aachen, RFA. (Refu le 22-5-1985, accept~ apr~s r(vision le 21-6-1985).
R6sum6 - - Le rdcepteur hlsulinique a dtd marqud h l'aide d'un ddrivd photor(actif de l'insuline (marquage spdcifique des sous-unitds ct) et par autophosphorylation en prdsence d'insuline (marquage spdcifique des sous-unitds fl). Les rdsultats montrent que le rdcepteur de l'insuline existe sous diffdrentes formes oligomdriques des sous-unitds ~t et fl lides entre elles par des ponts disulfure. De plus, le rdcepteur hTsulinique est dtroitement associd attx antigdnes de classe I du complexe majeur d'histocompatibilitd. Mots-cl~s : marquage par photoaffinit6 / tyrosine kinase / immnnopr~eipitation.
S u m m a r y - - Insulin receptors were labeled with 1251-photoreactive hlsulin (specifically labeling ct-subunits) and by insulin-sthnulated atttophosphorylation (specifically labeling fl-subunits). The results show that the hlsulin receptor exists under different free and disulfide-linked combinations of ct and fl subunits. Moreover, the insulin receptor is closely associated to class I antigens of the major histocompatibility complex to form a high molecular weight multi-molecular membrane complex. Key-words : photoaffinity labeling / tyrosine-kinase / immunoprecipitation.
Insulin, synthesized and secreted by pancreatic I~ cells, is involved at the level of its target cells in the regulation of a considerable number of metabolic and growth events. The first limiting step in the cellular responses to insulin is the recognition of the hormone by specific receptors expressed at the surface of the target cells. This binding step is followed by a large array o f biological effects taking place both at the plasma membrane level and at intracellular sites. The hormone-receptor interaction occurring at the cell surface has been characterized in detail [1, 2], but the mechanism of the hormonal signal transmis-
sion from the membrane to intracellular sites has not as yet been elucidated. Results accumulated during the last five years suggest that the clue to this problem resides in the structure of the insulin receptor itself. Indeed, antibodies specifically recognizing the insulin receptor are able to mimick most of the insulin biological effects, thus indicating that the information for hormone signalling is contained in the t-eceptor molecule and not in the ligand [3]. The detailed knowledge o f the structure of the insulin receptor thus seems to constitute a prerequisite to unravel the mechanism of insulin action.
1156
AL Fehlmann and coll.
Oiigomeric structure of the insulin receptor In most tissues and species investigated so far the insulin receptor is composed of at least 2 types of subunits linked together by disulfide bridges. The ct-subunit (Mr 130,000), which contains the insulin binding site, can be labeled by photoreactive insulin analogues [4, 5, 6] or with bifunctional crosslinking agents [7]. The [3-subunit (Mr 95,000) contains a tyrosine-kinase activity capable of autophosphorylation reactions in the presence of insulin [8, 9]. Thus by using these two biological properties of the insulin receptor, it is possible to selectively label the ct-subunits (by affinity labeling) and the [3-subunits (by autophosphorylation). When rat hepatoma cells (Fao) were incubated with ~'-5I-photoreactive insulin, only one protein (Mr 130,000) was labeled, as
shown in Figure 1 (lane C). This labeling was specific since it was inhibited by an excess of unlabeled insulin (Fig. 1, lane D). This protein corresponds to the ct-subunit of the hepatic insulin receptor containing the hormone binding site. When insulin receptors, partially purified by affinity chromatography, were incubated with (y-3'P)ATP and insulin, one major labeled protein (Mr 95,000) was observed (Fig. 1, lane B). This protein, which is not labeled in the absence .of insulin (Fig. 1, lane A) corresponds to the 13-subunit of the insulin receptor. When the labeling patterns obtained with mI-photoreactive insulin (ct-subunits) and (y-3Zp)ATP (fl-subunits) were compared after polyacrylamide gel electrophoresis under nonreducing conditions, the insulin receptor was found under different oligomeric forms (Fig. 2). These forms correspond to different combinations
32p 32 p e R ~
OR-- V
"° .
200--
94--
MrX
"'°'213
m
Ib,
116--
125 i
1251
0(2""
o( ----- I
." ~''
m
O--p
oo-
67~.
116--
43 -¸
94--
302014_=-- ..... 1() 3 A B
"" I O
O
67-m
dlBL C
D
I
M r × l O -3
A
B
C
D
FIG. I. -- Labeling of insufin receptors by autophosphoo'lation (J:P) amt with a photoreactive insufin analogue (~:~I).
FIG. 2. -- Labeling of insulin receptors by atttophosphorvlation (3:p) and with a photoreactive insulin analogue (t:~l)."
Insulin receptors were partially purified by affinity chromatography and phosphorylated with (yJ"P)ATP in the absence (A) or presence of 10-TM insulin (B). For photoaffinity labeling, cells were incubated with ':51-photoreactive insulin in the absence (C) or presence of 10-6M unlabeled insulin (D), UV-irradiated and directly solubilized in boiling SDS. Labeled proteins were analyzed by SDS-PAGE under reducing conditions using 7,5 % acrylamide gels, followed by autoradiography.
Insulin receptors were purified by lectin affinity chromatography and exposed to a control serum (A) or to a serum containing autoantibodies to the insulin receptor (B). After addition of protein A the immunoprecipitates were phosphorylated with (¥J:P)ATP. For photoaffinity labeling, cells were incubated with IZ~l-photoreactive insulin in the absence (t~) or presence of 10-6M unlabeled insulin (D), UV-irradiated and directly solubilized in boiling SDS. Labeled proteins were analyzed by SDS-PAGE under non-reducing conditions using 5 % acrylamide gels, followed by autoradiography.
Structure of insulin receptors of a- and 13-subunits : free ct, free 13, a13, a2, a213, and ct2 132 with apparent molecular masses of 95, 130, 220, 250, 350 and 450 kDa, respectively [10]. By contrast the I]2 combination was never found. It can thus be concluded that the insulin receptor is an immunoglobin-like heterotetramer, with 2 "heavy" chains (ct) linked together by disulfide bridges, each "heavy" chain being itself linked to a "light" chain (13) also by a S-S bridge (see Fig. 5). The presence of free insulin receptor subunits in native membranes of insulin-responsive cells has already been reported [11] but their contribution in receptor function and receptor regulation is not as yet totally understood. It seems, however, that oligomeric forms are preferentially subjected to receptor down-regulation when cells are exposed to high concentrations of insulin and that t h e number of free subunits is directly correlated to the average affinity of the receptor [11]. More complex models of the insulin receptor have been proposed on the basis of data obtained
i157
with photoreactive insulin analogues [12, 13]. Thus, the photoreactive analogue B29-Napa-insulin (the photoreactive residue being attached in position 29 of the insulin B chain) labels the ct-subunit (130 kDa) of the insulin receptor and in addition 4 proteins with molecular masses of 95, 85, 55 and 45 kDa (Fig. 3, lane C). The labeling of these proteins is specific (inhibited by unlabeled insulin : Fig. 3, lane D) and moreover, these proteins can be precipitated by anti-receptor antibodies. By contrast, the analogue B2-Napa-des PheBl-insulin labels only the u-subunit and a protein at 95 kDa (Fig. 3, lanes A and
B). It is therefore possible that in plasma membranes the insulin receptor is associated to other membrane proteins that can participate to the binding of insulin, since residue B29 is located in the insulin domain involved in the binding to the receptor.
Interactions with antigens of the major histocompatibility complex ( M H C ) A
B
C
D
E ....
OR-
F
200--
116--
-m,
9
94--
g
4
¢ 13o
67I
43--
tQ
tB
II
B1
'
II
B29
J
B2
FIG. 3. -- Labeling o f insulin receptors by different photoreactive analogues. Liver plasma membranes from C3H mice were incubated with BI-Napa-insulin (BI), B29-Napa-insulin (B29) and B2-Napa-des-Phe -8~ insulin (B2) (Napa : 2-nitro, 4-azidopbenylacetyl) in the absence (A, C, E) or in the presence (B, D, F) of 10 -6 unlabeled insulin, then UV-irradiated. Proteins were solubilized and analyzed as indicated in the legend to Figure 1.
Identification of membrane proteins associated with the insulin receptor can be undertaken by screening the effect of various anti-membrane protein antibodies on the binding of insulin to its receptor. Using this approach it has been demonstrated that insulin binding to HL60 cells (human promyelocytic cells) is inhibited by anti-HLA antibodies [14]. Figure 4 shows that insulin receptors from mouse liver plasma membranes, labeled with ~25I-photoreactive insulin, are precipitated by monoclonal antibodies reacting with H-2K (a heavy chain of class I antigens of the mouse MHC). Approximately 15% of the receptors which are precipitated by anti-receptor antibodies (Fig. 4, lane A) are precipitated by different anti H-2 antibodies (Fig. 4, lanes B-D), whereas no receptors are precipitated by an equivalent amount of normal mouse IgG's (Fig. 4, lane E) nor by a control serum (Fig. 4, lane F). This precipitation of insulin receptors by anti-H2 antibodies can be due to the presence of common epitopes on insulin receptors and H2 antigens (cross-reactivity) or to the formation of a molecular complex between those 2 proteins (coprecipitation). The experiment shown in Figure 4 indicates that 2 different monocional antibodies (la-
M. Fehlmann and coll.
1158 A
B
C
D
em
m
E
F
0R20011694-
O
/
¢ 13C
=,,
67--
43-ATP
30Mrxl() 3 ;
/
/
m- v 'as'
FIG. 4. -- hnnntnoprecipitation of insulin receptors by attti-H2 antibodies. Liver plasma membranes from CH3 (H-2~) mice were labeled with photoreactive insulin, solubilized in 1% Nonidet P-40 and incubated with : (A) serum containing anti-insulin receptor antibodies; (B) monoclonal antibodies reacting with Kk, D~ (H-100-30/23); (C) and (D) monoclonal antibodies reacting with Kk (H-100-5/28 and 11-4-1, respectively); (E) normal mouse IgG's; and (F) control serum. After addition of protein A, immunoprecipitates ~vereanalyzed by gel electrophoresis as indicated in the legend to Figure I.
nes B and C), known to react with distinct antigenic determinants [15, 16], are both capable of precipitating the receptor, which favors the coprecipitation possibility. Molecular association has been demonstrated by sequential immunoprecipitations (Fehlmann et al. submitted), and by using different mouse strains [17, 18]. These results thus show that in the hepatocyte plasma membrane the insulin receptor is closely linked to MHC antigens (Fig. 5). In the immune system MHC antigens are recognized by T lymphocytes in association with other membrane antigens. This process of corecognition is involved in the lysis of virus-infected cells by cytotoxic T cells (for class I MHC antigens) and in the stimulation of helper T cells by antigen presenting cells (for class II MHC antigens). The initial molecular event in this co-recognition process seems to reside in a physical interaction between MHC proteins and foreign antigens. Similarly, molecular interactions are believed to be involved in different non-
ADP
FIG. 5. -- The insulin receptor is associated to Class 1 (H-2K) antigens of the major histocompatibilio" complex in mouse liter membranes. immunological functions of M H C antigens including cell adhesion [20] and hormone action [21,22,23]. Anti-HLA antibodies have been shown to inhibit E G F [22] and insulin [14] binding to human cells in culture. Correspondingly, anti-fl2 microglobulin (the light chain o f class I MHC antigens) inhibits platelet aggregation induced by thrombin [23]. These results, together with the demonstration of a physical interaction between insulin receptors and class I M H C antigens, suggest that MHC antigens are directly involved in the transmission o f the hormonal signals triggered by the binding of hormones to their specific receptors. The key question is now the possible relationship between the kinase activity associated with the insulin receptor and MHC antigens. HLA antigens have been shown to be phosphorylated by the tyrosine-kinase associated with the Rous sarcoma virus [24] and similarly, preliminary results in our laboratory have shown that H-2 antigens are phosphorylated by the insulin receptor tyrosine-kinase. These observations may enable to reevaluate the biological functions o f the MHC, especially in the etiology of diabetes and in the immune tolerance of tumors that are under the control of growth and transformation factors.
Acknowledgments We are indebted to Anne-Marie Schmitt-Verhulst, Nicole Kiger, David Sachs and Ronald Kahn for helpful discussions and for the gift o f the different antibodies that have been used in this study.
Structure o f insulin receptors
I 159
REFERENCES 1. Kahn, C.R. (1976) J. Cell BioL. 70, 261-286. 2. Roth, J. (1979) In : Endocrinology (De Groot, ed.) Vol. 3, pp. 2037-2054, Grune and Stratton, New-York. 3. Kahn, C.R., Baird, K.L., Flier, J.S., Grunfeld, C., Harmon, J.T., Harrison, L.C., Karlsson, F.A., Kasuga, M., King, G., Lang, U.C., Podskalny, J.M. & Van Obberghen, E. (1981) Rec. Prog. Horm. Res., 37, 477-538. 4. Yip, C.C., Yeung, C.W.T. & Moule, M.L. (1980) Biochemistr)" 19, 70-76. 5. Wisher, M.H., Baron, M.D., Jones, R.H., S6nksen, P.H., Saunders, D.J., Thamm, P. & Brandenburg, D. (1980) Biochem. Biophys. Res. Commun., 92, 492-498. 6. Fehlmann, M., Le Marchand-Brustel, Y., Van Obberghen, E., Brandenburg, D. & Freychet, P. (1982) Diabetologia. 23, 440-444. 7. Pilch, P.F. & Czech, M.P. (1980) J. Biol. Chem., 25, 1722-1731. 8. Kasuga, M., Karlsson, F.A. & Kahn, C.R. (1982) Science, 215, 185-187. 9. Van Obberghen, E., Rossi, B., Kowalski, A., Gazzano, H. & Ponzio, G. (1983), Proe. Natl. Acad. Sci. USA, 80, 945-949. 10. Chvatchko, Y., Gazzano, H., Van Obberghen, E. & Fehlmann, M. (1984), Mol. Cell. Endocrinol., 36, 59-65.
il. Crettaz, M., Jialal, I., Kasuga, M. & Kahn, C.R. (1984) .L Biol. Chem., 259, 11543-11549. 12. Baron, M.D. & S6nksen, P.H. (1983) Biochem. J., 212, 79-84. 13. Yip, C.C. & Moule, M.L. (1983) Diabetes, 32, 760-767. 14. Simonsen, M. & Olsson, L. (1983) Ann. Immunol. (Paris), 134, 85-92. 15. Lemke, H., H~immerling, G.J. & H~mmerling, U. (1970) 1mmunol. Rev., 47, 175-182. 16. Oi, V.T., Jones, P.P., Goding, J.W. & Herzenberg, L.A. (1978) Curr. Top. MicrobioL Immunol., 81, 115-121. 17. Chvatchko, Y., Van Obberghen, E., Kiger, N. & Fehlmann M. (1983) FEBS Left., 163, 207-211. 18. Brossette, N., Van Obberghen, E. & Fehlmann, M. (1984) Diabetologia, 27, 74-76. 19. Edidin, M. (1983) Immunology Today 4, 269-270. 20. Curtis, A.S. & Rooney, P. (1979) Nature, 281, 222-223. 21. Lafuse, W. & Edidin, M. (1980) Biochemistry, 19, 49-54. 22. Schreiber, A.B., Schlessinger, J. & Edidin, M. (1984) J. Cell Biol., 98, 725-731. 23. Curry, R.A., Messner, R.P., Johnson, G.J. (1984) Science, 224, 509-511. 24. Guild, B., Erikson, R.L. & Strominger, J.L. (1983) Proc. NatL Acad. ScL USA, 80, 2894-2898.