Identification of Binding Partners of Fibroblast Gro wth Factor221 in Cell Membrane of 3T32L1 Cells

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology (7) : T32L1 FGF221 1), 1),, 3 (, ) (fibroblast growth factor,fgf)221 1, 2.,FGF221.,, 3T32L1, FGF221, 3T32L1., FGF kd., FGF221 FGF221 FGF221 ; FGF221,., FGF221., FGF221, ;, FGF mgπl,., FGF221 ; FGF221,., FGF SU5402 FGF221 3T32L1, FGF2212 FGF2212FGF. 221 (FGF221) ; ; Q71 Identification of Binding Partners of Fibroblast Gro wth Factor221 in Cell Membrane of 3T32L1 Cells WANG Wen2Fei 1), REN Gui2Ping 1), HOU Yu2Ting, LI De2Shan 3 ( College of Life Sciences, Northeast Agricultural University, Haerbin , China) Abstract Fibroblast growth factor ( FGF)221 is a novel regulator of insulin2independent glucose transport, and could be a potential therapeutic agent for treatment of type II diabetes. However, the precise mechanisms whereby FGF221 regulates blood glucose metabolism remain unknown. In order to study the function of FGF221 receptor, cross2link method was utilized ; 3T32L1 adipocytes were used as target cells and FGF221 was used as a bait to search for binding partners from 3T32L1 cell membrane. Biotinylated FGF221 could form two complexes with cell membrane proteins of 3T32L1 adipocytes. The size of the complexes was more than 300 kd. Competition experiment showed that the cold FGF221 could compete with labeled FGF221 in forming the complexes, the higher dose of the cold FGF221 was used, the stronger inhibition of the complex formation was observed. These results suggest that the complexes are FGF2212specific. In addition, the yield of the complexes increased with increment of the biotinylated FGF221 added to the reaction. However, the complexes became saturated when FGF221 concentration reached to 1215 mgπl or more. The result suggests that the formation of the complexes is FGF2212dependent. After the binding sites of the FGF2212associated proteins : ; : (No. 2006G ) 3 Tel : ; E2mail : com 1), Received :January 3,2008 ;Accepted :March 13, 2008 Supported by Science and Technology Planning Program of Heilongjiang Province (No. 2006G ) 3 Corresponding author Tel : ; E2mail : com 1) WANG Wen2Fei and REN Gui2Ping made equal contribution to the paper

2 were saturated, the quantity of complexes reached a maximum. Furthermore, FGF receptor inhibitor SU5402 could restrain FGF2212induced glucose up2take in 3T32L1 adipocytes, suggesting that the complexes might be the FGF2212FGF receptor. Key words fibroblast growth factor221 ( FGF221) ; adipocytes ; complex identification (fibroblast growth factor, FGF) (bfgf) 65 [1 ]. FGF [2 ].,, FGF , FGF. 1 FGF223,,. 2 FGF221. FGF221 [3 ], 2005, Kharitonenkov [4 ], FGF221, 2.,., FGF221,,,. FGF221,. FGF221,,, [5 ],,, [6 ]. FGF221,. FGF,FGF221,, [4 ]., FGF221 3T32L1,. FGF FGF,, FGF221 FGF ( )., FGF221 FGF, FGF221. 3T32L1 [7 ],.,FGF221 3T32L1, 3T32L1 [3 ], 3T32L1 FGF221., 3T32L1, FGF221, FGF FGF221 FGF221 cdna, Pet30ΠBL21 (DE3), C Flag His.. Flag (Sigma) FGF2212Flag, Ni2NTA (Qiagen) FGF2212His. Waters. HPLC 95 % 112 3T32L1 3T32L1, 10 % DMEM 3T32L1 ( ). 3T32L1, 1 (5 mgπl,0125 molπl,015 mmolπl IBMX,10 % DMEM ) 2 d, 2 (5 mgπl,10 % DMEM ) 2 d, 10 % DMEM, 2 d 1, d, 90 % T32L Π 96, 4 d. 14 d,, KRPH (15 mmolπl Hepes, 118 mmolπl NaCl, 418 mmolπl KCl, 112 mmolπl MgSO 4, 113 mmolπl CaCl 2, 112 mmolπl KH 2 PO 4, 011 % BSA, ph 714) FGF221, FGF h, KRPH 2, KRPH 100 l [ 100 molπl 14 C 22 2D2 ( ) ] h, Cytochlasin B ( 20 molπl, Sigma). ( 262 ).

3 7 :3T32L1 FGF FGF221,, FGF221, FGF221 BaF3,,., 2 BaF3 FGF221., FGF221 ( ) min,. 12 % ( 25 mmolπl Tris,250 mmolπl Gly (ph 813) ). 230 ma 3 h,, 5 % 1 h. 4, PBS2T 3, 5 min, HRP 1 h, PBS2T 3, PBS 3, 5 min. SuperSignal ( Pierce), Hyperfilm (Amersham). 116 (Cat. No : 21336, Pierce Biotechnology Inc. Rockford, IL). FGF biotin2nhs. 2 h, biotin2nhs. FGF221, FGF T3L h, FGF2212Flag FGF221, 4 2 h. Hanks 2., BS 3, 4 2 h., 50 mmolπl Tris,150 mmolπl NaCl ph 715,37 15 min. (50 mmolπl Tris, 1 % Triton X2100, 011 % Chaps, 150 mmolπl NaCl, 1 mmolπl EDTA) (Roche, Indianapolis, IN). FGF221Π. : FGF221, streptavidin2 (Sigma) ; Flag FGF221, Flag ( Sigma). IP SDS2 PAGE, FGF221. FGF221, FGF221 FGF221Π. 118 FGF FGF 32[ ( 32( 22Carboxyethyl )242methylpyrrol222yl ) methylene ]222indolinone (SU5402, Calbiochem) FGF [8 ]. FGF221Π, FGF FGF221, 50 molπl SU5402 3T32L1 30 min, FGF221, SU5402, 24 h, SU5402, T32L1 3T32L1 4 d,. 6 d,,, 14 d 90 %. Fig. 1 3T32L1 14 d. 212 FGF221 FGF221Π FGF2212Flag 3T3L1, BS 3., Triton X2100 NP40,, Flag, SDS2PAGE, FGF221 FGF221 (Fig. 2A FGF2 21 ) kd.. FGF221.,FGF221,, FGF221, ( Fig. 2A)., FGF221. FGF2212Biotin 3T3L1, BS 3.

4 670 24, Triton X2100,, Streptavidin2, SDS2PAGE, FGF221 2 ( Fig. 2B)., FGF221 (Fig. 2B). Fig. 1 The differentiation of 3T32L1 cells in vitro. (A) The morphogenesis of 3T32L1 cells before differentiations under low power. (B) The morphogenesis of fully differentiated adipocytes under low power (100 ) Fig. 2 FGF221Πmembrance protein complexes in adipocytes (A) Adipocytes were incubated with Flag2tagged FGF221 and followed by incubation with BS 3, cells were lysed after the cross2link reagent quenched. The cell lysate was immunoprecipitated with anti2flag antibody beads ( Sigma). The complexes were analyzed in SDS2PAGE and immunoblotted with anti2fgf221 polyclonal antibodies. 1 : Adipocytes without treatment. 2 : Adipocytes + FGF2212Flag (1 mgπl) + BS 3,lysed with NP401 3 : Adipocytes + FGF2212Flag (10 mgπl) + BS 3,lysed with NP40. 4 : Adipocytes + FGF2212Flag (10 mgπl) without BS 3,lysed with NP40. 5 : Adipocytes + FGF2212Flag (10 mgπl) + BS 3,lysed with Triton X : Pre2adipocytes + FGF2212Flag (10 mgπl) + BS 3,lysed with TritonX : Adipocytes BS 3 only, lysed with TritonX2100. (B) Adipopcytes were incubated with biotinylated FGF221 and followed by incubation with BS 3, cells were lysed after the cross2link reagent quenched. The cell lysate was immunoprecipitated with streptavidin affinity gel ( Sigma). complexes were analyzed in SDS2PAGE and immunoblotted with anti2fgf221 polyclonal (1) or monoclonal (2) antibodies. Adipocytes without treatment lysed as control The 2. 3 FGF221 FGF221 FGF221, FGF221., FGF221 FGF221. FGF221 1 h, FGF221, FGF221., FGF221 FGF221Π,. FGF221,100 FGF221 (Fig. 3).,, FGF221. FGF221

5 7 :3T32L1 FGF , (BSA).,BSA FGF221. Fig. 3 The cold FGF221 blocked the biotinylated FGF221 to form the complexes (A) For competition, 1, 10 and 100 fold excess of cold FGF221 proteins were added to the cell surface of 182day2old 3T3L1 adipocytes together with 1 mgπl biotinylated FGF221. The complexes were cross2linked using non2reducing cross2link reagent BS 3 as described in Materials and Methods. The complexes were immunoprecipitated with streptavidin2agarose beads and Western2detected with rabbit anti2human FGF221 antibody. (B) To eliminate the non2specific inhibition of exogenous protein, we did the same experiment with BSA as a control. The complexes were not inhibited by BSA at any concentration 214 FGF221 FGF221, FGF221., FGF2212 Biotin, Streptavidin2 Biotin2FGF221Π, FGF221., FGF221 ( Fig. 5), FGF mgπl, ( ).,., 250 kd FGF FGF SU5402 FGF2 21 3T32L1, 3T32L1 FGF221, FGF221,. FGF2 21Π 2, Fig. 4 The complexes were FGF221 dose dependent Cell surface of 182day2old 3T32L1 adipocytes were cross2linked with different amounts of biotinylated FGF221 ( mgπl) using non2reducing cross2link reagent BS 3 as described in Materials and Methods. The complexes were immunoprecpitated with streptavidin2agarose beads and Western2detected with rabbit anti2fgf221 antibody 250 kd, 2, 250 kd,2

6 kd., 100 kd, FGF. FGF, FGF FGF221. FGF SU5402, FGF221 FGF,SU5402 FGF2 21.,3T32L1 50 molπl SU min, FGF221, SU5402, 24 h, SU5402..,,FGF221,. FGF, ( Fig. 5). 50 molπl SU5402, ( ). Fig. 5 FGF221 activity was inhibited by FGFR inhibitor SU5402 3T32L1 adipocytes were pretreated with 50 molπl SU5402 for 30 min prior to addition of FGF221. The cells were further treated with the same concentration of SU5402 for 24 hours in the presence of FGF221. The glucose uptake by these cells was measured as described in Materials and Methods. The data showed that treatment of adipocytes with this inhibitor abolished FGF2212induced glucose2uptake. The values ( SE) shown are the average of 3 independent measurements. 3 P < 0105 as compared with cells that were not treated with SU FGF,,. FGF221 FGF,. FGF221 [9 ]. FGF221,FGF221 FGF. FGF221 3T3L1. FGF221,,,. FGF2 21.,3T3L1 FGF221.,FGF221 FGF , 3T3L1, FGF221,,,., FGF2 21Π.,..,FGF kd Π,. FGF221. FGF221,1215 mgπl (FGF221), FGF221.. FGF FGF [10 ], FGF221, 100 kd, FGF. FGF, FGF FGF221.,FGF FGF221,. FGF221Π FGF. FGF,. FGF221,. FGF221 ( PPARs)

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