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A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.

MLTC-1 cells, derived from a murine Leydig tumor, contain a gonadotropin-responsive adenylate cyclase that became desensitized to hCG. Prior exposure to hCG reduced the ability of MLTC-1 cells to accumulate cAMP by approximately 50%, but caused only a small reduction in hCG receptor number. Membranes isolated from desensitized cells showed a similar reduction in hCG-stimulated adenylate cyclase activity. Desensitization was time, temperature, and dose dependent. Elevating intracellular cAMP levels by incubating the cells with (Bu)2cAMP or cholera toxin failed to cause desensitization. Desensitization did not depend on protein synthesis. Desensitization caused no change in the dose response of adenylate cyclase to hCG or GTP. hCG receptor affinity for hCG was not affected by desensitization or guanine nucleotides. The stimulatory regulatory component of adenylate cyclase (Ns) from MLTC-1 cells was used to reconstitute S49 cyc- membranes, which lack Ns. Ns from control and desensitized MLTC-1 cells were equally effective in reconstitution of the beta-adrenergic-sensitive adenylate cyclase of cyc-. beta-Adrenergic receptors from cyc- membranes were also transferred to MLTC-1 membranes by fusion with polyethylene glycol to produce a beta-adrenergic-responsive adenylate cyclase. Isoproterenol-stimulated activity was similar, regardless of whether membranes from control or desensitized MLTC-1 cells were used. We conclude that neither Ns nor the catalytic subunit of the adenylate cyclase in MLTC-1 cells is the site of lesion in desensitization. Most likely, the hCG receptor itself may be affected when MLTC-1 cells are desensitized by hCG.

The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.

The murine Leydig tumor cell line, MLTC-1, contains a gonadotropin receptor-coupled adenylate cyclase. Although the binding of human choriogonadotropin (hCG) initially causes cells to accumulate cAMP, in time, the response to hCG is attenuated by desensitization. Treating intact cells with the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or with diacylglycerol also causes desensitization of the hCG response. These compounds are activators of calcium/phospholipid-dependent protein kinase (PKC). Treating MLTC-1 cells with TPA or dioctanoylglycerol increased the portion of PKC in the cell membrane fraction. This phenomenon is associated with activation of PKC. Treating isolated membranes with purified PKC desensitize the hCG response. Thus, desensitization caused by TPA or dioctanoylglycerol is probably mediated by PKC. PKC is normally activated when phosphoinositides are metabolized to diacylglycerol and inositol phosphates. There was no significant accumulation of inositol phosphates when cells were treated with hCG. hCG did not increase the portion of PKC in the cell membrane fraction. However, hCG could desensitize isolated membranes, but TPA could not. We conclude that although protein kinase C activity can desensitize the gonadotropin response, hCG does not cause desensitization by activating PKC. The implications of this observation are discussed.

The murine Leydig tumor cell line, MLTC-1, has specific cell surface receptors for human chorionic gonadotropin (hCG), which are coupled to adenylate cyclase. When the cells were exposed to hCG, there was a loss of these receptors (down-regulation), which was both dose- and time-dependent. Down-regulation was inhibited by lowering the temperature, removing bound hormone or blocking protein synthesis with cycloheximide. Down-regulation was found to be a biphasic event. The initial phase was dependent upon the binding of hormone and had a half-time of approximately 3 h. The second phase occurred around 8 h after exposing the cells to the hormone and was apparently independent of bound hCG. It was related to increases in cyclic AMP since it could be mimicked by incubating the cells with choleragen or dibutyryl cyclic AMP and isobutylmethylxanthine. The reappearance of hormone receptors began approximately 24-32 h after the initial exposure to hCG and was complete within 48 h. Adenylate cyclase activity in membranes from control and down-regulated cells responded equally well to Mn2+ and NaF indicating that neither the catalytic or regulatory component (G/F) of the adenylate cyclase system had been lost during downregulation. Cholate extracts of control and down-regulated cells also were equally effective at reconstituting isoproterenol-stimulated adenylate cyclase activity in S49 cyc-membranes (which lack a functional G/F). Thus, down-regulation did not impair the ability of G/F to couple receptors to the catalytic component of adenylate cyclase.

The murine Leydig tumor cell line 1 (MLTC-1) contains gonadotropin receptors (GR) that are coupled to adenylate cyclase through the stimulatory guanine nucleotide binding protein (Gs). The binding of human choriogonadotropin (hCG) causes MLTC-1 cells to accumulate cAMP. With time, the ability of MLTC-1 cells to respond to hCG is attenuated by a process called desensitization. The hydrodynamic properties of GR from control and desensitized MLTC-1 cells were studied. Sucrose density gradient sedimentation in H2O and D2O and gel filtration chromatography were used to estimate the Stokes radius (a), partial specific volume (vc), sedimentation coefficient (S20,w), and molecular weight (Mr) of the detergent-solubilized hormone-receptor complex (hCG-GR). [125I]hCG was bound to MLTC-1 cells under conditions that allow (37 degrees C) or prevent (0 degree C) desensitization, and hCG-GR was solubilized in Triton X-100. In the absence of desensitization, control hCG-GR had a Mr of 213,000 (a = 6.2; vc = 0.76; S20,w = 7.3), whereas desensitized hCG-GR had a Mr of 158,000 (a = 6.1; Vc = 0.71; S20,w = 6.6). Deglycosylated hCG (DG-hCG) is an antagonist that binds to GR with high affinity but fails to stimulate adenylate cyclase or cause desensitization. [125I]DG-hCG was bound to MLTC-1 cells and DG-hCG-GR solubilized in Triton X-100. The hydrodynamic properties of DG-hCG-GR (Mr 213,000; a = 5.8; Vc = 0.77; S20;w = 7.6) were the same as that for control hCG-GR. There was no evidence for the association of adenylate cyclase or Gs with GR in Triton X-100 solubilized preparations.(ABSTRACT TRUNCATED AT 250 WORDS)