Insulin-like growth factor binding protein-3 (IGFBP-3) can inhibit cell growth by directly interacting with cells, as well as by forming complexes with IGF-I and IGF-II that prevent their growth-promoting activity. The present study examines the mechanism of inhibition of DNA synthesis by IGFBP-3 in CCL64 mink lung epithelial cells. DNA synthesis was measured by the incorporation of 5-bromo-2'-deoxyuridine, using an immunocolorimetric assay. Recombinant human IGFBP-3 (rh[N109D,N172D]IGFBP-3) inhibited DNA synthesis in proliferating and quiescent CCL64 cells. Inhibition was abolished by co-incubation of IGFBP-3 with a 20% molar excess of Leu(60)-IGF-I, a biologically inactive IGF-I analogue that binds to IGFBP-3 but not to IGF-I receptors. DNA synthesis was not inhibited by incubation with a preformed 1:1 molar complex of Leu(60)-IGF-I and IGFBP-3, indicating that only free IGFBP-3 inhibits CCL64 DNA synthesis. Inhibition by IGFBP-3 is not due to the formation of biologically inactive complexes with free IGF, since endogenous IGFs could not be detected in CCL64 conditioned media; any IGFs that might have been present could only have existed in inactive complexes, since endogenous IGFBPs were present in excess; and biologically active IGFs were not displaced from endogenous IGFBP complexes by Leu(60)-IGF-I. After incubation with CCL64 cells, (125)I-IGFBP-3 was covalently cross-linked to a major thick similar400-kDa complex. This complex co-migrated with a complex formed after incubation with (125)I-labeled transforming growth factor-beta (TGF-beta) that has been designated the type V TGF-beta receptor. (125)I-IGFBP-3 binding to the thick similar400-kDa receptor was inhibited by co-incubation with unlabeled IGF-I or Leu(60)-IGF-I. The ability of Leu(60)-IGF-I to decrease both the inhibition of DNA synthesis by IGFBP-3 and IGFBP-3 binding to the thick similar400-kDa receptor is consistent with the hypothesis that the thick similar400-kDa IGFBP-3 receptor mediates the inhibition of CCL64 DNA synthesis by IGFBP-3.

An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein {SU, residues 1 to 236 [SU:(1-236)]} and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (K d , 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain. MATERIALS AND METHODS Construction of expression plasmids encoding MCAT-1 and Friend SU. The vector pSP64T (16) was modified by (i) insertion of the BamHI-to-XbaI portion of the polylinker from pCDNA3 (Invitrogen) at the BglII/SalI site, (ii) reinsertion of the deleted BglII-to-SmaI fragment of pSP64T at the BamHI/PvuII site and addition of a second polylinker containing NsiI, BglII, ApaI, and PstI sites at the PstI site, and (iii) insertion of a 140-bp fragment encoding (in tandem) a factor Xa protease site (amino acids IEGR), three copies of the hemagglutinin (HA) peptide (YPYDVPYA [10]) recognized by monoclonal antibody 12CA5 (Boehringer Mannheim), and a six-histidine motif at the XhoI/PstI site in the polylinker (Flu 3-his 6 tag). The new vector was designated pRD67:Flu 3-his 6. A clone encoding envelope SU of Friend MLV (reference 12; GenBank accession no. J02192) in plasmid pUC13 (a gift from A. Pinter) was modified by addition of a 5Ј EcoRI site at bp Ϫ40 relative to the initiator ATG and a 3Ј XhoI site at bp 810 that permitted insertion into pRD67:Flu 3-his 6 with an in frame fusion at the 3Ј end to the sequences encoding the protease cleavage site and Flu 3-his 6 tag. A cDNA encoding the ecotropic retrovirus receptor (MCAT-1) with the Cterminal protease cleavage site and Flu 3-his 6 tag (MCAT-1t) was constructed by using PCR to place an EcoRI site at bp Ϫ50 relative to the initiator ATG and to replace the stop codon of MCAT-1 (residue 622) with an in-frame XhoI recognition site and inserted into the polylinker of pRD67:Flu 3-his 6. To avert the possibility of PCR-induced mutations, the BamHI-to-SphI fragment of this plasmid was excised and replaced by the equivalent fragment from the original MCAT-1 cDNA clone. The absence of Taq polymerase-induced changes in the remaining portion (Ͻ10% of the coding region) was confirmed by nucleotide sequencing. Expression of recombinant proteins in oocytes and mammalian cells. Capped mRNAs encoding MCAT-1 or MCAT-1t were transcribed and injected into Xenopus laevis oocytes, and after 3 days, transport of L-[ 14 C]arginine and exogenous binding of 125 I-SU proteins were measured as described elsewhere (11). In some experiments, detergent extracts of oocytes were prepared and solubilized proteins separa...

The relationship between endocrine axes is not simple. An improved understanding of the interactions between neuroendocrine systems may facilitate the design of efficient drug regimens in the treatment of diseases such as osteoporosis and muscle wasting.