Faculty Bibliography

Melanocyte-stimulating hormone (MSH) induces melanogenesis in Cloudman mouse melanoma cells. The activities of two enzymes in the melanogenesis pathway, tyrosinase and dopachrome conversion factor, are increased as part of the induction process. Trans retinoic acid (RA), at concentrations as low as 0.1 nM, inhibited the induction of tyrosinase, dopachrome conversion factor, and melanogenesis, but had no effect on the basal levels of either enzyme or of cellular melanin content. Half-maximal effects of RA occurred at a concentration of 10 nM; maximal effects were observed at 1 microM. The effects of RA on melanogenesis were independent of its effects on cellular growth since one Cloudman line tested was growth-inhibited by RA and another was growth-stimulated by RA, but the induction of melanogenesis by MSH in both lines was inhibited by RA. Mixing experiments with cell lysates failed to demonstrate the induction of a tyrosinase inhibitor by RA. The effects of RA were not limited to MSH or to Cloudman melanoma cells since RA blocked cholera toxin-inducible melanogenesis in Cloudman cells, as well as the induction of tyrosinase activity by L-tyrosine in Bomirski hamster melanoma cells. The effects of RA were specific to melanogenesis, however, since RA did not interfere with MSH-induced changes in cellular morphology and growth. Thus, RA appears to be a new and potent tool for understanding mechanisms regulating induction of the pigmentary system

Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient (8-80%) and centrifuged (156,000g, 60 min); fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: 1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. 2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. 3) Most of the internal binding sites were not as dense as fully melanized melanosomes. 4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. 5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) 6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone

The murine macrophage-like cell line J774.16 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin (CaM)-binding protein (CaMBP) which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and peritoneal macrophages elicited with concanavalin A, lipopolysaccharide, proteose peptone, or Bacillus Calmette-Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein, as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed with the use of a rabbit antiserum raised to the partially purified CaM-binding protein and [125I]CaM covalently cross-linked to the principal CaM-binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein, suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared, and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of Mr 50,000 to 60,000 was isolated. The protein could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property, plus the sensitivity of the protein to endoglycosidase F, led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization, and evidence for glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for Ca2+-CaM

A calmodulin-binding protein is present in extracts of the macrophage-like mouse cell line J774 and in extracts of thioglycollate-stimulated mouse peritoneal macrophages; it is deficient in variants of J774 resistant to trifluoperazine and in resident peritoneal macrophages. The calmodulin-binding protein [CaMBP (J7)0.5] was purified from J774 and resolved from endogenous cyclic nucleotide phosphodiesterase and protein kinase activities. The protein has an apparent native Mr of 125,000-150,000 and binds calmodulin in a calcium-dependent manner with a Kd of 20 nM. It inhibits the ability of calmodulin to activate phosphodiesterase. Its sedimentation constant in glycerol gradients containing calmodulin was dependent upon the relative concentrations of calmodulin and the calmodulin-binding protein

Two forms of the extracellular cyclic nucleotide phosphodiesterase (EC 3.1.4.17) of Dictyostelium discoideum have been purified. One species has a molecular weight of about 55,000 measured by gel filtration and has been purified to apparent homogeneity. This monomeric form of the enzyme can be resolved by isoelectrofocusing into 4 bands with isoelectric points between 7.5 and 9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands with molecular weights of 50,000 and 48,000. The second form of the enzyme has been partially purified and has a higher molecular weight (150,000-200,000) and an isoelectric point of about 5. The high molecular weight form of the enzyme is converted to the low molecular weight monomer by isoelectrofocusing in the presence of 6 M urea. Under these conditions the isoelectric point is shifted to that of the monomeric form of the enzyme. The two species are immunologically indistinguishable after urea treatment, and react identically with the cyclic nucleotide phosphodiesterase inhibitor (see the accompanying paper: Franke, J., and Kessin, R. H. (1981) J. Biol. Chem. 256, 7628-7637). The two forms have the same Km and show equal sensitivity to a number of ions, chelators, and low molecular weight cyclic nucleotide phosphodiesterase inhibitors. Tryptic maps of the purified monomeric enzyme and the urea dissociated high molecular weight form revealed no differences

The extracellular adenosine 3',5'-cyclic monophosphate phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) produced by Dictyostelium discoideum has two kinetic forms. The free enzyme has a Km of approximately 10 microM. The second form is the result of a complex formed with a heat-stable inhibitor and has a Km in the millimolar range. Treating the enzyme-inhibitor complex with dithiothreitol stimulated enzyme activity 20- to 100-fold and changed in Km from millimolar to micromolar. Dithiothreitol inactivated the inhibitor. Reconstituting purified enzyme with excess inhibitor returned the Km to the millimolar range. Under conditions known to inhibit the production of extracellular inhibitor or in mutants that lack it, extracellular phosphodiesterase activity was already high and could not be increased by dithiothreitol. The phosphodiesterase and inhibitor sedimented at 6 S and 3 S, respectively; the enzyme-inhibitor complex sedimented at 6.7 S