The cDNAs encoding three major insulin-like growth factor-binding proteins (IGFBPs) have been cloned and sequenced. We have examined, by Western ligand blotting, the profiles of these binding proteins in human female serum in the normal menstrual cycle, throughout pregnancy, and during the postpartum period. There was no change in the serum profile of any of the binding proteins in early pregnancy compared to that in the secretory phase of the menstrual cycle. However, there was a marked decrease in circulating levels of the main serum IGFBP, IGFBP-3, after 6 weeks of gestation, continuing progressively to term and returning to nonpregnant levels by 5 days postpartum. IGFBP-2 decreased steadily throughout gestation. In contrast, IGFBP-1 levels were found to rise by the second trimester. Endoglycosidase-F digestion did not enhance detection of IGFBP-3 by ligand blotting. Immunoprecipitations with two separate antibodies against IGFBP-3 and IGFBP-2, followed by Western ligand blotting, confirmed the marked decrease in IGFBP-3 levels after 6 weeks of gestation and the more gradual decrease in IGFBP-2. In contrast, immunoprecipitations with IGFBP-1 monoclonal antibodies confirmed the increase in IGFBP-1 during gestation. Endogenous serum IGFs were separated from serum IGFBPs by acid chromatography, and an 80% decrease in total IGF-binding activity in the IGFBP fraction of chromatographed pregnancy vs. nonpregnancy serum was detected by charcoal absorption assay. Furthermore, immunoprecipitations of IGF affinity cross-linked IGFBP fractions with IGFBP-3-specific antiserum confirmed a marked diminution of IGFBP-3 in pregnancy compared to nonpregnancy serum, and revealed, only in pregnancy serum, the concomitant appearance of a band with a mol wt of 34K and three less intense bands with mol wt between 20-26K on sodium dodecyl sulfate gels. Incubation of nonpregnancy serum with 6-week pregnancy serum at 37 C for 5 h, followed by Western ligand blotting, showed only a slight reduction in the amount of IGFBP-3 in the mixture compared to that in controls. However, incubation of term pregnancy with nonpregnancy serum at 37 C for 5 h revealed a marked reduction of IGFBP-3 in the mixture. When iodinated recombinant IGFBP-3 was incubated with term pregnancy serum under the same conditions, the appearance of a 29K protein was identified by gel electrophoresis and autoradiography, along with three less intense bands with mol wt between 17-22K.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factors (IGFs) are potent mitogens that bind with high affinity and specificity to IGF receptors and IGF-binding proteins (IGFBPs). We studied the roles of these three groups of proteins in prostate epithelial cells (PEC) in primary culture grown under serum-free conditions. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from PEC revealed an abundance of type 1 IGF receptors and no evidence of type 2 IGF receptors. Western ligand blots of conditioned media (CM) from PEC demonstrated the presence of two specific IGFBP bands similar to those previously demonstrated in seminal plasma, with approximate mol wt of 31 and 24 kDa. The 31-kDa band was immunoprecipitable with an antibody to IGFBP-2, and neither band could be deglycosylated with endoglycosidase-F. Northern blot analysis of poly(A)+ RNA prepared from PEC with cDNAs for hIGFBP-1, -2, and -3 documented the expression of mRNA for hIGFBP-2 only. Modifications of the serum-free conditions of PEC did not significantly alter the IGFBP profile of PEC CM. The ability of IGF-I, IGF-II, and insulin to stimulate clonal growth of PEC was examined. IGF-I stimulated PEC growth with an ED50 of 0.1 ng/mL. IGF-II and insulin, respectively, were 1 and 3 orders of magnitude less effective than IGF-I in stimulating the growth of PEC. Radioimmunoassayable IGF-I and IGF-II levels in PEC CM were below the assay detection levels. In conclusion, we suggest that IGFs are important growth stimulators of PEC in culture, that their actions are mediated through the type 1 IGF receptor, and that PEC produce hIGFBP-2 and a 24-kDa IGFBP which may modulate IGF action in these cells.

An immunocompetent B lymphocyte can be triggered by the appropriate antigen and lymphokine signals to differentiate into a pentamer IgM-secreting ceil. The process involves a change in the function of the IgM antibody molecule; it is converted from a membrane receptor for antigen to a secreted polymer that effects antigen disposal. This change is known to require (a) synthesis of the secreted form of # heavy chain and (b) synthesis of the polymerizing component, the Ig J chain.Membrane and secreted forms of the # heavy chain were first distinguished by their different mobilities in polyacrylamide gels (1). Subsequent studies showed that the structural differences are confined to the carboxy-terminal sequence of the chain and correlate with the different functions of the IgM molecule (2, 3). The membrane form has a hydrophobic terminus that anchors the IgM monomer in the B cell membrane whereas the secreted form has a shorter, more hydrophilic terminus that contains the penultimate cysteine residue required for pentamer IgM assembly. The two termini were found to be encoded in exons located 3' to the C# gene and are cotranscribed with the gene (4). Separate ~m and ~ts mRNAs are then generated from the single gene transcript by differential 3' cleavage and processing. These data established that the switch in ~ chain synthesis during B cell differentiation is regulated at the posttranscriptional level.The assembly and secretion of pentamer IgM also depends on the synthesis of a second protein, the J chain (5, 6). The J polypeptide serves to initiate IgM polymerization by bridging two IgM monomers through disulfide bonds to the #s carboxy termini. The resulting J chain-containing dimer then promotes the disulfide bonding of three additional monomers to complete the pentamer structure. Studies of rabbit and human splenocytes (7,8) have shown thatJ chain expression is a direct result of B cell activation. Little or no intracellular J chain could be detected in the unstimulated cell populations. After stimulation, however, large amounts of J chain were observed concomitant with the appearance of secreted IgM in the culture supernatants. Moreover, analyses of J chain mRNA in transformed cell lines showed that J chain-specific sequences were absent from a line representing an unstimulated B cell, but were present in large amounts in a myeloma cell line secreting pentamer IgM (9). These results indicate

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