CLEAVAGE OF MEMBRANE-ASSOCIATED PREF-1 GENERATES A SOLUBLE INHIBITOR OF ADIPOCYTE DIFFERENTIATION

pref-1 is an epidermal growth factor-like repeat protein present on th e surface of preadipocytes that functions in the maintenance of the pr eadipose state. pref-1 expression is completely abolished during 3T3-L 1 adipocyte differentiation. Bypassing this downregulation by constitu tive expression of full-length transmembrane pref-1 in preadipocytes d rastically inhibits differentiation. For the first time, we show proce ssing of cell-associated pref-1 to generate both a soluble pref-1 prot ein of approximately 50 kDa that corresponds to the ectodomain and als o smaller products of 24 to 25 kDa and 31 kDa. Furthermore, while all four of the alternately spliced forms of pref-1 produce cell-associate d protein, only the two largest of the four alternately spliced isofor ms undergo cleavage in the juxtamembrane region to release the soluble 50-kDa ectodomain. We demonstrate that addition of Escherichia coli-e xpressed pref-1 ectodomain to 3T3-L1 preadipocytes blocks differentiat ion, thus overriding the adipogenic actions of dexamethasone and methy lisobutylxanthine. The inhibitory effects of the pref-1 ectodomain are blocked by preincubation of the protein with pref-1 antibody. That th e ectodomain alone is sufficient for inhibition demonstrates that tran smembrane pref-1 can be processed to generate an inhibitory soluble fo rm, thereby greatly extending its range of action. Furthermore, we pre sent evidence that alternate splicing is the mechanism that governs th e production of transmembrane versus soluble pref-1, thereby determini ng the mode of action, juxtacrine or paracrine, of the pref-1 protein.