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Mol Cell Biol. 1997 February; 17(2): 977–988.
PMCID: PMC231823

Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation.

Abstract

pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes that functions in the maintenance of the preadipose state. pref-1 expression is completely abolished during 3T3-L1 adipocyte differentiation. Bypassing this downregulation by constitutive expression of full-length transmembrane pref-1 in preadipocytes drastically inhibits differentiation. For the first time, we show processing of cell-associated pref-1 to generate both a soluble pref-1 protein of approximately 50 kDa that corresponds to the ectodomain and also smaller products of 24 to 25 kDa and 31 kDa. Furthermore, while all four of the alternately spliced forms of pref-1 produce cell-associated protein, only the two largest of the four alternately spliced isoforms undergo cleavage in the juxtamembrane region to release the soluble 50-kDa ectodomain. We demonstrate that addition of Escherichia coli-expressed pref-1 ectodomain to 3T3-L1 preadipocytes blocks differentiation, thus overriding the adipogenic actions of dexamethasone and methylisobutylxanthine. The inhibitory effects of the pref-1 ectodomain are blocked by preincubation of the protein with pref-1 antibody. That the ectodomain alone is sufficient for inhibition demonstrates that transmembrane pref-1 can be processed to generate an inhibitory soluble form, thereby greatly extending its range of action. Furthermore, we present evidence that alternate splicing is the mechanism that governs the production of transmembrane versus soluble pref-1, thereby determining the mode of action, juxtacrine or paracrine, of the pref-1 protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Appella E, Weber IT, Blasi F. Structure and function of epidermal growth factor-like regions in proteins. FEBS Lett. 1988 Apr 11;231(1):1–4. [PubMed]
  • Brachmann R, Lindquist PB, Nagashima M, Kohr W, Lipari T, Napier M, Derynck R. Transmembrane TGF-alpha precursors activate EGF/TGF-alpha receptors. Cell. 1989 Feb 24;56(4):691–700. [PubMed]
  • Breyer JA, Cohen S. The epidermal growth factor precursor isolated from murine kidney membranes. Chemical characterization and biological properties. J Biol Chem. 1990 Sep 25;265(27):16564–16570. [PubMed]
  • Buxbaum JD, Gandy SE, Cicchetti P, Ehrlich ME, Czernik AJ, Fracasso RP, Ramabhadran TV, Unterbeck AJ, Greengard P. Processing of Alzheimer beta/A4 amyloid precursor protein: modulation by agents that regulate protein phosphorylation. Proc Natl Acad Sci U S A. 1990 Aug;87(15):6003–6006. [PubMed]
  • Carpenter G, Cohen S. Epidermal growth factor. J Biol Chem. 1990 May 15;265(14):7709–7712. [PubMed]
  • Cheng HJ, Flanagan JG. Transmembrane kit ligand cleavage does not require a signal in the cytoplasmic domain and occurs at a site dependent on spacing from the membrane. Mol Biol Cell. 1994 Sep;5(9):943–953. [PMC free article] [PubMed]
  • Christy RJ, Yang VW, Ntambi JM, Geiman DE, Landschulz WH, Friedman AD, Nakabeppu Y, Kelly TJ, Lane MD. Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes. Genes Dev. 1989 Sep;3(9):1323–1335. [PubMed]
  • Evan GI, Lewis GK, Ramsay G, Bishop JM. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. [PMC free article] [PubMed]
  • Faust IM, Johnson PR, Stern JS, Hirsch J. Diet-induced adipocyte number increase in adult rats: a new model of obesity. Am J Physiol. 1978 Sep;235(3):E279–E286. [PubMed]
  • Fehon RG, Kooh PJ, Rebay I, Regan CL, Xu T, Muskavitch MA, Artavanis-Tsakonas S. Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila. Cell. 1990 May 4;61(3):523–534. [PubMed]
  • Flanagan JG, Chan DC, Leder P. Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant. Cell. 1991 Mar 8;64(5):1025–1035. [PubMed]
  • Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell. 1995 Dec 1;83(5):803–812. [PubMed]
  • Green H, Kehinde O. Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell. 1976 Jan;7(1):105–113. [PubMed]
  • Green H, Kehinde O. Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line. J Cell Physiol. 1979 Oct;101(1):169–171. [PubMed]
  • Gullberg U, Lantz M, Lindvall L, Olsson I, Himmler A. Involvement of an Asn/Val cleavage site in the production of a soluble form of a human tumor necrosis factor (TNF) receptor. Site-directed mutagenesis of a putative cleavage site in the p55 TNF receptor chain. Eur J Cell Biol. 1992 Aug;58(2):307–312. [PubMed]
  • Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM. Weight-reducing effects of the plasma protein encoded by the obese gene. Science. 1995 Jul 28;269(5223):543–546. [PubMed]
  • Hatsuzawa K, Murakami K, Nakayama K. Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites. J Biochem. 1992 Mar;111(3):296–301. [PubMed]
  • Herrera R, Ro HS, Robinson GS, Xanthopoulos KG, Spiegelman BM. A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation. Mol Cell Biol. 1989 Dec;9(12):5331–5339. [PMC free article] [PubMed]
  • Hu E, Tontonoz P, Spiegelman BM. Transdifferentiation of myoblasts by the adipogenic transcription factors PPAR gamma and C/EBP alpha. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9856–9860. [PubMed]
  • Huang EJ, Nocka KH, Buck J, Besmer P. Differential expression and processing of two cell associated forms of the kit-ligand: KL-1 and KL-2. Mol Biol Cell. 1992 Mar;3(3):349–362. [PMC free article] [PubMed]
  • Ibrahimi A, Bonino F, Bardon S, Ailhaud G, Dani C. Essential role of collagens for terminal differentiation of preadipocytes. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1314–1322. [PubMed]
  • Jensen CH, Krogh TN, Højrup P, Clausen PP, Skjødt K, Larsson LI, Enghild JJ, Teisner B. Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2. Eur J Biochem. 1994 Oct 1;225(1):83–92. [PubMed]
  • Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell. 1995 Dec 1;83(5):813–819. [PubMed]
  • Klyde BJ, Hirsch J. Increased cellular proliferation in adipose tissue of adult rats fed a high-fat diet. J Lipid Res. 1979 Aug;20(6):705–715. [PubMed]
  • Klyde BJ, Hirsch J. Isotopic labeling of DNA in rat adipose tissue: evidence for proliferating cells associated with mature adipocytes. J Lipid Res. 1979 Aug;20(6):691–704. [PubMed]
  • Kohno T, Brewer MT, Baker SL, Schwartz PE, King MW, Hale KK, Squires CH, Thompson RC, Vannice JL. A second tumor necrosis factor receptor gene product can shed a naturally occurring tumor necrosis factor inhibitor. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8331–8335. [PubMed]
  • Kopczynski CC, Alton AK, Fechtel K, Kooh PJ, Muskavitch MA. Delta, a Drosophila neurogenic gene, is transcriptionally complex and encodes a protein related to blood coagulation factors and epidermal growth factor of vertebrates. Genes Dev. 1988 Dec;2(12B):1723–1735. [PubMed]
  • Laborda J, Sausville EA, Hoffman T, Notario V. dlk, a putative mammalian homeotic gene differentially expressed in small cell lung carcinoma and neuroendocrine tumor cell line. J Biol Chem. 1993 Feb 25;268(6):3817–3820. [PubMed]
  • Luetteke NC, Michalopoulos GK, Teixidó J, Gilmore R, Massagué J, Lee DC. Characterization of high molecular weight transforming growth factor alpha produced by rat hepatocellular carcinoma cells. Biochemistry. 1988 Aug 23;27(17):6487–6494. [PubMed]
  • Massagué J. Transforming growth factor-alpha. A model for membrane-anchored growth factors. J Biol Chem. 1990 Dec 15;265(35):21393–21396. [PubMed]
  • Massagué J, Pandiella A. Membrane-anchored growth factors. Annu Rev Biochem. 1993;62:515–541. [PubMed]
  • Mroczkowski B, Reich M, Chen K, Bell GI, Cohen S. Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity. Mol Cell Biol. 1989 Jul;9(7):2771–2778. [PMC free article] [PubMed]
  • Pandiella A, Bosenberg MW, Huang EJ, Besmer P, Massagué J. Cleavage of membrane-anchored growth factors involves distinct protease activities regulated through common mechanisms. J Biol Chem. 1992 Nov 25;267(33):24028–24033. [PubMed]
  • Parries G, Chen K, Misono KS, Cohen S. The human urinary epidermal growth factor (EGF) precursor. Isolation of a biologically active 160-kilodalton heparin-binding pro-EGF with a truncated carboxyl terminus. J Biol Chem. 1995 Nov 17;270(46):27954–27960. [PubMed]
  • Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 1995 Jul 28;269(5223):540–543. [PubMed]
  • Rall LB, Scott J, Bell GI, Crawford RJ, Penschow JD, Niall HD, Coghlan JP. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature. 1985 Jan 17;313(5999):228–231. [PubMed]
  • Ray P, Moy FJ, Montelione GT, Liu JF, Narang SA, Scheraga HA, Wu R. Structure-function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene. Biochemistry. 1988 Sep 20;27(19):7289–7295. [PubMed]
  • Rubin CS, Hirsch A, Fung C, Rosen OM. Development of hormone receptors and hormonal responsiveness in vitro. Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells. J Biol Chem. 1978 Oct 25;253(20):7570–7578. [PubMed]
  • Smas CM, Green D, Sul HS. Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1. Biochemistry. 1994 Aug 9;33(31):9257–9265. [PubMed]
  • Smas CM, Sul HS. Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell. 1993 May 21;73(4):725–734. [PubMed]
  • Smas CM, Sul HS. Control of adipocyte differentiation. Biochem J. 1995 Aug 1;309(Pt 3):697–710. [PubMed]
  • Spiegelman BM, Ginty CA. Fibronectin modulation of cell shape and lipogenic gene expression in 3T3-adipocytes. Cell. 1983 Dec;35(3 Pt 2):657–666. [PubMed]
  • Teixidó J, Massagué J. Structural properties of a soluble bioactive precursor for transforming growth factor-alpha. J Biol Chem. 1988 Mar 15;263(8):3924–3929. [PubMed]
  • Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 1994 May 15;8(10):1224–1234. [PubMed]
  • Wharton KA, Johansen KM, Xu T, Artavanis-Tsakonas S. Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell. 1985 Dec;43(3 Pt 2):567–581. [PubMed]
  • Wong ST, Winchell LF, McCune BK, Earp HS, Teixidó J, Massagué J, Herman B, Lee DC. The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction. Cell. 1989 Feb 10;56(3):495–506. [PubMed]
  • Wu Z, Xie Y, Bucher NL, Farmer SR. Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis. Genes Dev. 1995 Oct 1;9(19):2350–2363. [PubMed]
  • Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994 Dec 1;372(6505):425–432. [PubMed]

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