Journal of IVeuroc/ie,ni,itrr Li ppincott—Raven PuhI ishers, Philadelphia 1996 International Society I or Neurochemistry Insulin-Like Growth Factor I Receptor Expression and Function in Nerve Growth Factor—Differentiated PC12 Cells Eugenia Yakovchenko, Michael Whalin, Vilen Movsesyan, and Gordon Guroff Section on Growth Factors, National Institute of Child Health and Hwnan Development, National Institutes of Health, Bethesda, Man/and, U.S.A. Abstract: Receptors for insulin-like growth factor I (IGF- I) were studied on PC12EY cells, a subclone of PC12. Differentiation of PC12EY cells with nerve growth factor (NGF) did not alter either the number of IGF-l receptors nor their affinity for IGF-I. IGF-I receptors remained fully functional during differentiation, promoting increases in thymidine incorporation, glucose uptake, amino acid up- take, and the phosphorylation of the S6 protein of the ribosomes. IGF-I also increased the proportion of differ- entiated cells found in S-phase. But although the addition of IGF-l to naive cells caused an increase in cell number, there was no comparable increase when IGF-I was added to differentiated cells. Thus, although the receptor for IGF-I continues to be present and functional, IGF-l fails to induce cell proliferation in differentiated P012 cells. Key Words: P012 Insulin-like growth factor— Nerve growth factor. J. Neurochem. 67, 540—548 (1996). The insulin-like growth factors (IGFs) are a family of proteins with both growth-promoting and insulin- like activity. Several lines of evidence indicate that the IGFs may serve as autocrine/paracrine growth factors in the developing nervous system. IGF transcripts are abundant in fetal and adult brain (Lund et al., 1986; Bondy and Lee, 1993). In addition, receptors for the IGFs are widely distributed in neural tissue (Lesniak et al., 1988; LeRoith et al., 1993). In vitro, the IGFs act on several different neural cells (DiCiccio-Bloom and Black, 1988; McMorris and Dubois-Dalcq, 1988; Masters and Raizada, 1993). Nerve growth factor (NGF) was the first and is the best-characterized member of a family of polypeptides now known as the neurotrophins. NGF is a survival factor in vivo and in neuronal cell culture as well, and appears to promote the differentiation of several classes of neurons (Levi-Montalcini and Angeletti, 1968; Levi-Montalcini, 1987). Information regarding the actions of NGF on several cell types is readily available, but the precise molecular mechanism(s) of NGF-initiated signal transduction is still poorly under- stood and is just now being clarified. PCI2 cells, a clonal line isolated from a rat adrenal medullary tumor, have proved useful asa model sys- tem for studying the action of NGF (Greene and Tischler, 1976; Dichter et al., 1977). When cultured in the presence of NGF, PCI2 cells exhibit the hall- marks of sympathetic neuronal differentiation, which include the slowing or cessation of cell division, the acquisition of excitable properties, the activation of neurotransmitter biosynthetic pathways, and the elabo- ration of neurites. Among other things, NGF treatment causes down-regulation of epidermal growth factor (EGF) receptors (Huff et al., 1981 ). As EGF has been shown to he a mitogen for PCI2 cells, it has been suggested that NGF differentiation of PCI2 cells may be due, at least in part, to the down-regulation of recep- tors for mitogens such as EGF. Insulin-like growth factor I (IGF-I) is also a mitogen for PCI2 cells (Dahrner and Perlman, 1988; Nielsen and Gammeltoft, 1988), and IGF-I receptors have pre- viously been shown to be present on these cells (Dahmer et al.. 1989). The purpose of this study was to determine if NGF-induced differentiation of PCI 2 cells alters the expression and/or the function(s) of the IGF-I receptor. To study the characteristics of the IGF-I receptor on PC12EY cells, a cell line cloned from PC 12, radioligand binding studies, affinity label- ing and cross-linking, and immunoblot analysis were performed using control PCI2EY cells and PCI2EY cells treated with NGF. IGF-l receptor function was examined by testing the ability of IGF-I to stimulate Received January 25, 1996: revised manuscript received March 19, 996; accepted March 2!. 1996. Address correspondence and reprint requests to Dr. G. Guroff at Section on Growth Factors. National Institute of Child Health and Human Developrnetst. Building 49. Room 5A64, Bethesda, MD 20892, U.S.A. Abbret’ialio,t.s used: BSA. bovine serum albumin: DMEM. Dul- hecco’s modified Eagle’s medium: DTT. dithiothreitol: EGF. epider- mal growth factor: IGF, insulin-like growth factor; IGF-l. IGF type I; LSM, low serum medium: MTT, 3—(4.5-dimethylthiazol—2—yl 2.5-diphenyllelrai.oliurn bromide; NGF. nerve growth factor: NSM. normal serum medium; PAGE, polyacrylamide gel eleetrophoresis: PBS. phosphate-buFfered saline: PMSF. phenylmelhylsulfonyl Ilito- ride: SDS. sodium dodecyl sulfate: TCA. trichloroacetic acid. 540