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The expression of melanocortin-5 receptor (MC5-R) mRNA and protein was characterized from isolated rat lymphocytes. The presence of MC5-R mRNA in spleen and thymus tissues was demonstrated by RT-PCR. The RT-PCR product was sequenced to confirm the identification of MC5-R. Tissues from lachrymal glands, adipose, adrenals, thymus, pancreas, and isolated splenic lymphocytes were detergent solubilized. The crude proteins were resolved by SDS-PAGE, transblotted to a nitrocellulose membrane, and probed for MC5-R using anti-receptor rabbit antisera. Two different types of polyclonal rabbit antisera were raised against synthetic peptides representing epitopes found at the amino (alphaN-MC5-R) and the carboxyl termini (alphaC-MC5-R) on the MC5-R. A prominent band at 77,000 (p77) was detected in all tissues except the pancreas. Preimmune sera did not detect p77 by Western analysis and the addition of peptide antigen neutralized the detection of p77 by the specific antisera. The receptor protein was purified from spleen and thymic lymphocytes using protein A agarose that precipitated material complexed to alphaN-MC5-R. The purified MC5-R was detected by Western analysis using alphaC-MC5-R. Both anti-receptor antisera, alphaN-MC5-R and alphaC-MC5-R, detected the p77. The p77 was treated with protein endoglycosidase F to produce a smaller protein band between 34-38,000 (p35); the inferred size is 37,000 based on the cDNA sequence. The data suggest that Asn-linked carbohydrate groups account for much of the p77 mass of the MC5-R. The data also demonstrate the expression of MC5-R protein on rat lymphocytes, thus, supporting the hypothesis that MC5-R is the ACTH receptor on lymphocytes.

The effect of incubation temperature and ligand competition was tested for (125)I-ACTH binding to isolated rat lymphocytes. AlphaMSH but not Agouti-like peptide was an effective competitive inhibitor for cell surface binding at 4 degrees C. Cells incubated with (125)I-ACTH at 37 degrees C rapidly associated ligand for 10 min and then gradually lost the radioactivity with time. Cells incubated with (125)I-ACTH at 4 degrees C accumulated ligand to only about half the maximal amount when compared to cells incubated at 37 degrees C for 10 min. Temperatures below 20 degrees C and toxins that block lysosomal degradation blocked the loss of cell-associated radioactivity. These results suggest the lymphocyte ACTH receptor is the Melanocortin 5 receptor and the receptor is internalized by endocytosis to deliver ligand to the lysosome.

Freshly isolated rat lymphocytes were tested for corticotropin (ACTH)-dependent calcium uptake. Physiological levels of corticotropin (0.01-1 nM) were found to stimulate both an uptake of 45Ca2+ and a rise in cAMP. The calcium uptake was delayed by 2 min after ACTH addition, but was rapid and transient after the onset of uptake. The extent of calcium uptake was dose dependent on the corticotropin concentration and reached a maximum by 1 nM. Several fragments of corticotropin were tested for activity; both full-length 1-39 and a functional truncated form, 1-25, had equivalent effects on 45Ca influx at 1 nM; however, alpha MSH-(1-13), ACTH-(11-24), or a mixture of alpha MSH and ACTH-(11-24) had no effect on 45Ca influx. Extracellular calcium uptake was blocked by the calcium channel blockers lanthanum, diltiazem, nifedipine, and omega-conotoxin. Splenic lymphocytes that express ACTH receptors had ligand-dependent calcium uptake, but thymocytes that lack ACTH receptors had no ligand-dependent calcium uptake. A mouse adrenal cell line, Y-1, showed the same 45Ca uptake kinetics. These findings demonstrate that both lymphocytes and adrenal cells have a functional ACTH-dependent calcium uptake mechanism.

The molecular recognition theory predicts that a reversed (3'----5') reading of an mRNA should yield a peptide that is structurally and functionally similar to that specified in the 5'----3' direction. We tested this idea by synthesizing a corticotropin (ACTH) analogue using a reverse reading of bovine mRNA for ACTH-(1-24). This peptide, designated ACTH-3'----5', had a similar hydropathic profile to native ACTH-5'----3' but had only 30% sequence homology and eight different charge substitutions. ACTH-3'----5' specifically bound to the surface of mouse Y-1 adrenal cells and to polyclonal anti-ACTH antibody. Additionally, ACTH-3'----5' stimulated cAMP synthesis and steroidogenesis in adrenal cells. These findings show that ACTH-3'----5' mimics the corticotropic properties of native ACTH, thereby further validating the molecular recognition theory.

We have produced a monoclonal antibody that specifically recognizes the adrenocorticotropic receptor on rat adrenal cells. The immunogen was designed from an RNA sequence complementary to the mRNA coding for ACTH1–24. This complementary peptide, termed HTCA, has been shown to specifically bind ACTH and was proposed to mimic the ACTH binding site of the hormone receptor. The monoclonal anti-HTCA antibody recognized a restricted domain of the HTCA peptide, bound to Y-1 adrenal cells with a KD of 1.8 nM, and blocked the binding of 125I-ACTH to rat adrenal cells. These findings show that anti-HTCA competes with ACTH for binding to the ACTH receptor.

The initiation of DNA synthesis and secretion of Interleukin 2 (IL-2) was measured in isolated rat splenic lymphocytes following activation with Concanavalin A (ConA). The extent of 3H-thymidine incorporation into activated cells was tested when cultured with various concentrations of Adrenocorticotropic hormone (ACTH). A paradoxical dose-response curve resulted when ACTH caused a biphasic response of augmenting and inhibiting 3H-thymidine uptake in lymphocytes depending on the hormone concentration. Low levels of ACTH (0.001-1-nM) augmented 3H-thymidine uptake and high levels (10-1000 nM) reversed the effect. The optimal ACTH concentration was 10 pM ACTH in the presence of 5 ug/ml ConA and there was no ACTH effect on quiescent cells (no ConA). Conditioned media from splenic lymphocytes treated with various concentrations of ConA or ACTH was tested for increased uptake of 3H-thymidine by the IL-2 growth dependent Cytotoxic T Lymphocyte Leukemia (CTLL-2) cells. ConA conditioned medium could sustain the CTLL-2 cells indicating the presence of IL-2. Conditioned medium from splenic lymphocytes treated with both ConA and 100 pM ACTH further increased CTLL-2 cell proliferation indicating an additional increase of IL-2 secretion. The identity of IL-2 was confirmed by using an anti-rat IL-2 antibody to neutralize the growth potential of the conditioned medium. ACTH alone had no effect on the CTLL-2 cell proliferation indicating the effect is due solely to induced IL-2 found in the conditioned medium. IL-2 levels in the conditioned media were quantitated by ELISA assay; splenic lymphocytes produced 4.2 ng/ml to ConA only, 19.2 ng/ml in ConA plus 10 nM ACTH, and no detectable IL-2 at ConA plus 10 uM ACTH. These results demonstrated that ACTH modulates IL-2 secretion from activated lymphocytes, which is both biphasic and concentration dependent.

This paper reviews a method for the design of peptides and proteins of predefined structure and function and provides an example. Specifically, an analog of rat growth hormone-releasing hormone (GHRH) (residues 1-23) was synthesized by solid-phase methods based on a reversed sequence of the mRNA for GHRH (1-23). The new peptide, designated GHRH 3′-5′, had a hydropathic profile similar to that of native GHRH 5′-3′ (GHRH) but had only 17% primary sequence homology. GHRH 3′-5′ specifically bound to the GHRH receptor on rat pituitary cells and to polyclonal anti-GHRH antibody in ELISA and RIA procedures. Additionally, GHRH 3′-5′ blocked the in vitro stimulation of GH RNA synthesis and in vitro and in vivo GH release mediated by GHRH. These data show that 3′-5′ GHRH with little sequence homology to native rat GHRH is an antagonist and further supports the importance of the linear pattern of hydropathy to the gross secondary and/or tertiary structure and rudimentary function of peptides and proteins. The impact of these findings on the interaction of complementary peptides is discussed.

We demonstrate that mitogen-activated lymphocytes release a biologically active form of ACTH that stimulates the in vitro release of corticosterone from cocultured rat adrenal cells. Neither nonstimulated lymphocytes nor the addition of mitogens alone to adrenal cell cultures had an effect. The steroidogenic activity could be neutralized by rabbit anti-ACTH serum, but not by a nonimmune serum. Both Concanavalin-A- and lipopolysaccharide-stimulated lymphocytes secrete an ACTH-like molecule with an antigenic specificity identical to pituitary-derived ACTH. Further, the amount of measurable immunoreactive ACTH was far lower than the amount of exogenously added ACTH required to evoke such a vigorous glucocorticoid response, suggesting that local deposition of the hormone results in a higher effective ACTH concentration. In addition, lymphocytes physically isolated from adrenal cells by a semipermeable membrane could stimulate steroidogenesis by 48 h, which corresponds to the rise in ACTH detected by RIA. These results confirm that activated lymphocytes synthesize as well as release biologically active ACTH, thus providing an in vitro model for a bidirectional communication between the endocrine and immune systems.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.

A monoclonal antibody that specifically recognizes the adrenocorticotropic receptor (ACTH) on rat adrenal cells was tested for hormonal activity. The antibody behaved as an agonist based on three different biological activities associated with ACTH. An antibody concentration of 16 micrograms/ml stimulated isolated rat adrenal cells to secrete 800 ng/10(4) cells of corticosterone with a concomitant 10-fold increase of cAMP to 30 pmol/10(5) cells. Antibody concentrations above 16 micrograms/ml inhibited mitotic activity in mouse Y-1 adrenal cells. A radio-immunoassay using an anti-ACTH antibody showed that the monoclonal anti-adrenocorticotropic receptor antibody and ACTH are antigenically related. These findings suggest that the anti-receptor antibody recognizes the ligand binding domain of the ACTH receptor.

After internalization of 125I-asialo-orosomucoid (ASOR) by isolated rat hepatocytes, ligand dissociates by two kinetically distinct pathways (Oka and Weigel, J. Biol. Chem. 257, 10,253, 1983). These slow and fast dissociation pathways correspond to two functionally different subpopulations of cell surface galactosyl receptors designated, respectively, State 1 and State 2 receptors. Freshly isolated cells or cells equilibrated below 24 degrees C express only State 1 receptors. Cells equilibrated at 37 degrees C express both State 1 and State 2 receptors. Ligand dissociation after internalization of surface-bound 125I-ASOR was measured using the permeabilizing detergent, digitonin. The slow dissociation pathway was mediated by State 1 receptors and was the only pathway expressed by cells which were freshly isolated or had been equilibrated at 24 degrees C. State 2 receptors are expressed at temperatures above about 20 degrees C, and both the fast and slow dissociation pathways occurred in cells equilibrated at 37 degrees C. State 2 receptors therefore mediate the rapid dissociation pathway. Dissociation and subsequent degradation of specifically bound ligand routed in either pathway were complete, respectively, within 3 and 6 hrs.

Factors affecting the synthesis of mannosylphosphoryl dolichol and glucosylphosphoryl dolichol hen oviduct microsomes were compared in order to gain insight into the properties of their respective synthases. A stabilized form of mannosylphosphoryl dolichol synthase, but not glucosylphosphoryl dolichol synthase, was released from microsomes by freezing the membranes after exposure to the detergent CHAPSO. The activation energy for mannosylphosphoryl dolichol synthesis in membranes was 9.4 kcal/mol over the range of 10–37°C; above 18°C the process of glucosylphosphoryl dolichol synthesis in membranes had a similar activation energy, 8.1 kcal/mol, but below 18°C the value was 16.7 kcal/mol. Tryptic digestion of sealed microsomes preferentially inactivated mannosylphosphoryl dolichol synthase; however, both synthases were equally inactivated in detergent-permeabilized microsomes. Periodate-oxidized UDP-Glc was used to probe the topological orientation of glucosylphosphoryl dolichol synthase in rat liver microsomes. Sealed microsomes treated with oxidized UDP-Glc were inactive in synthesis of glycosylphosphoryl dolichol. However, when these treated microsomes were permeabilized, glucosylphosphoryl dolichol synthase activity was readily detected. From these studies we conclude that although mannosyl- and glucosylphosphoryl dolichol synthases catalyze chemically similar reactions in the endoplasmic reticulum, they differ in several respects. These differences were interpreted in terms of a topological model in which the active sites of the two enzymes reside on opposite faces of the endoplasmic reticulum, with that of the glucosyl lipid synthase facing the lumen and that of the mannosyl lipid synthase facing the cytosol.

We have shown that degradation of asialo-orosomucoid (ASOR) in isolated rat hepatocytes occurs by two different intracellular pathways [Clarke, Oka & Weigel (1987) J. Biol. Chem. 262, 17384-17392] mediated by two subpopulations of cell surface galactosyl (Gal) receptors, designated State 1 or State 2 receptors. In the present study, several inhibitors were tested for their effects on ligand degradation by the State 1 or State 2 pathway. Leupeptin, monensin and chloroquine completely inhibited degradation of 125I-labelled ASOR in both pathways. Dose-response studies showed, however, that the State 2 pathway was more sensitive to leupeptin or monensin than the State 1 pathway. No differences were observed with chloroquine. For example, the onset of inhibition in the State 2 and State 1 pathways occurred at about 0.05 and 0.3 microM-leupeptin respectively, a 6-fold difference. At 3.5 microM-monensin, 125I-ASOR degradation in the State 2 pathway was completely blocked, whereas degradation in the State 1 pathway was essentially unaffected. Colchicine was observed to give the largest differential sensitivity between the two pathways. The State 2 degradation pathway was about 30-fold more sensitive to colchicine than the State 1 pathway. Lumicolchicine had no affect. The onset of inhibition of the rate of 125I-ASOR degradation in the State 2 and State 1 pathways occurred at approximately 0.1 and 3.0 microM-colchicine respectively. At very high concentrations (greater than 0.1 mM), the State 1 pathway could be completely inhibited. We conclude that intracellular 125I-ASOR processing or delivery to degradative compartments in both the State 1 and State 2 Gal receptor pathways requires low pH. Ligand delivery to the degradative compartment does not require microtubules in the State 1 pathway, consistent with the very rapid onset of degradation in this pathway. The State 2 degradation pathway does require microtubules.