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Heterotrimeric (alphabetagamma) G(s) mediates agonist-induced stimulation of adenylyl cyclase (AC). Cholera toxin (CTx) will ADP-ribosylate the alpha-subunit of G(s) (G(s)alpha). G(s)alpha-deficient cyc(-) membranes were "stripped" of Gbeta. When the stripped cyc(-) were incubated with G(s)alpha and/or Gbetagamma, each was incorporated into the membranes independently of the other. Both G(s)alpha and Gbetagamma had to be present in the membranes, and they had to be able to form a heterotrimer in order for CTx to ADP-ribosylate G(s)alpha, indicating that the membrane bound G(s) heterotrimer is a substrate for CTx, but the G(s)alpha subunit by itself is not. When G(s)alpha was completely and irreversibly activated with GTPgammaS and incorporated into stripped cyc(-), it was a poor substrate for CTx and a weak stimulator of AC unless Gbetagamma was also incorporated. Furthermore, the level of AC stimulation corresponded to the amount of G(s) heterotrimer that was formed in the membranes from GTPgammaS-activated G(s)alpha and Gbetagamma. These data suggest that AC is stimulated by an activated G(s) heterotrimer in cell membranes.

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Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 microliters) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots of S20,w versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated alpha-subunit were determined. The results were similar to those obtained with 17- to 22-h centrifugations in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.

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Heterotrimeric (alpha beta gamma) G proteins mediate a variety of signal transduction events in virtually every cell of every eukaryotic organism. The predominant hypothesis is that dissociation of the alpha-subunit from the G beta gamma-subunit complex necessarily accompanies the activation of these proteins, and that the alpha-subunit is primarily responsible for regulating the response of effector molecules. However, there is increasing evidence that both the alpha-subunit and the beta gamma-subunit complex function in regulating effector activity. Furthermore, data for some G proteins suggest that they function as activated heterotrimers rather than as dissociated subunits.

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The stimulatory guanine nucleotide binding protein (Gs) is heterotrimeric ( alpha beta gamma), and mediates activation of adenylyl cyclase by a ligand-receptor complex. The alpha subunit of Gs (Gsalpha) has a guanine nucleotide binding site, and activation occurs when tightly bound GDP is displaced by GTP. Together, GDP and fluoroaluminate (AlF4-) form a transition state analog of GTP that activates Gs. The work of other investigators suggests that AlF4- causes subunit dissociation when it activates Gs. We have observed that in solution AlF4- did not cause Gs subunits to dissociate unless NaCl was also present. The effect of NaCl was concentration dependent (10-200 mM). Omitting F-, Al3+, or Mg2+ prevented the NaCl-induced dissociation of Gs subunits. Na2SO4 could not substitute for NaCl in causing subunit dissociation, but KCl could, suggesting that the anion was responsible for the effect. Gs subunit reassociation occurred when the concentration of Cl- was reduced even though the concentrations of AlF4- and Mg2+ were maintained. The absence of Cl- did not prevent AlF4- binding to Gsalpha. We have concluded that AlF4-, a ligand which is capable of activating G proteins, can bind to Gs in solution without causing subunit dissociation.

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The stimulatory G protein (Gs) mediates activation of adenylyl cyclase. Gs is a heterotrimeric protein (alpha beta gamma) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue displaces tightly bound guanosine diphosphate (GDP) from the guanine nucleotide-binding site of the alpha-subunit (Gs alpha). Divalent cations such as magnesium are also required for Gs activation. Subunit dissociation can accompany Gs activation and is thought to be critical for this process. We investigated the effects of MgCl2 and various purine nucleotides on Gs-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein beta-subunit that was co-precipitated by Gs alpha-specific antiserum. Gs activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc-membranes that lack Gs alpha. High concentrations of MgCl2 caused bound GDP to dissociate from Gs and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl2 caused a concentration-dependent dissociation of Gs subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to lower concentrations of MgCl2, suggesting that GTP gamma S promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl2. These results suggest that GTP, the compound that activates Gs in vivo, was no more effective at promoting Gs subunit dissociation than was GDP.

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Human choriogonadotropin (hCG) is a glycoprotein hormone that activates adenylyl cyclase. The carbohydrate moieties of hCG are required for biological activity, but not for binding to the gonadotropin receptors. We modified N-acetylneuraminic acid (NeuAc) on the oligosaccharide moieties of hCG, and determined the effect on its biological activity by measuring hormone-stimulated adenylyl cyclase. Treating hCG with sodium periodate to remove two carbon atoms from NeuAc or quantitatively removing NeuAc from hCG reduced its biological activity by 36% and 50%, respectively. The galactose residues of asialo-hCG were reacted with NeuAc-hydrazone or a hydrazone of the oligosaccharide from the ganglioside GM1 (Gal(beta 1-3)GalNAc(beta 1-4) [NeuAc(alpha 2-3)]Gal(beta 1-4)Glc). The gonadotropin receptor had high affinity for both derivatives, but their biological activity was less than that of hCG. These results suggest that several structural aspects of NeuAc including carbon side chain, an intact ring structure, and the position of NeuAc relative to other carbohydrate residues are important for full biological activity of hCG.

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The stimulatory G protein (Gs) mediates activation of adenylylcyclase by a ligand-receptor complex. Gs is heterotrimeric (alpha beta gamma) and activation can be accomplished by dissociation of the alpha-subunit (Gs alpha) from the beta gamma-subunit complex (G beta gamma). Gs alpha is also a substrate for choleragen catalyzed ADP-ribosylation when it is associated with G beta gamma but not as free Gs alpha. Using recombinant DNA techniques we modified the cDNA for the 52,000 M(r) form of Gs alpha (Gs alpha 52) to produce a protein with a 2,400 M(r) N-terminal extension (Gs alpha 54.4). This N-terminal extension could be removed with the protease Factor Xa. In vitro transcription and translation of the recombinant plasmid containing the cDNA's for Gs alpha 52 and Gs alpha 54.4 produced a 52,000 M(r) and a 54,000 M(r) protein, respectively. In solution the properties of Gs alpha 52 and Gs alpha 54.4 were indistinguishable. Both proteins: (a) formed a heterotrimer with G beta gamma and their affinities for the subunit complex were the same; (b) could be ADP-ribosylated by choleragen in the presence but not in the absence of G beta gamma; (c) bound the non-hydrolyzable GTP analogue, GTP gamma S, and were protected from chymotryptic proteolysis by the guanine nucleotide; and (d) could activate in vitro translated type IV adenylylcyclase. Gs alpha 54.4 and Gs alpha 52 were incorporated into S49 cyc-membranes, which lack Gs alpha. After incorporation, both Gs alpha 52 and Gs alpha 54.4 were protected from chymotryptic proteolysis when GTP gamma S was present, revealing that both proteins were able to bind the nucleotide and undergo a conformational change characteristic of Gs alpha activation. When Gs alpha 52 was incorporated into cyc-membranes it could mediate both hormone and GTP gamma S stimulation of adenylylcyclase and could be ADP-ribosylated by choleragen, but Gs alpha 54.4 could do neither of these things, indicating that the properties of Gs alpha 54.4 were altered by the membrane. Deletion of the N-terminal extension by treatment with Factor Xa in solution converted Gs alpha 54.4 to Gs alpha 52, and upon incorporation into cyc-membranes it behaved like Gs alpha 52 in every regard, showing that the effect of the N-terminal extension was reversible. A lack of other differences in the functional properties of Gs alpha 52 and Gs alpha 54.4 suggests a correlation between the interaction of Gs alpha with G beta gamma and its ability to activate adenylylcyclase.

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Type IV adenylyl cyclase was synthesized in a cell-free coupled transcription and translation system. Radiolabeled type IV adenylyl cyclase was specifically immune precipitated with anti-ACIV antibodies. The molecular weight of in vitro translated type IV adenylyl cyclase was 110,000, similar to that for type IV adenylyl cyclase produced in the baculovirus system [B. Gao and A. G. Gilman, (1991) Proc. Natl. Acad. Sci. USA 88, 10178-10182]. Dimyristoyl phosphatidylcholine was required for efficient stimulation of activity by both forskolin and GS, with a maximum specific activity of 700 +/- 100 nmol cAMP.min-1.mg-1 attained with both effectors combined. Both bovine brain GS and in vitro translated GS alpha activated in vitro translated type IV adenylyl cyclase; however, G beta gamma only enhanced stimulation in the presence of in vitro translated GS alpha. Forskolin maximally activated at concentrations from 200 to 400 microM in the absence or presence of GS. The in vitro translated product was very stable as production of cAMP by forskolin/GS activated type IV adenylyl cyclase was linear for up to 90 min at 30 degrees C.

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The heterotrimeric (alpha beta gamma) stimulatory G protein (Gs) mediates activation of adenylylcyclase. Gs is inactive when GDP is bound to the guanine nucleotide binding site of the alpha-subunit (Gs alpha). Gs can be activated by fluoroaluminate or by binding GTP or GTP analogues, (e.g., GTP gamma S) in place of GDP. Magnesium ion is also required for the activation of Gs, and Gs alpha is a substrate for ADP-ribosylation catalyzed by choleragen (CT). Gs activation can also be accompanied by dissociation of Gs alpha from the G beta gamma-subunit complex. When dissociated Gs subunits were separated by chromatography, isolated Gs alpha could not be ADP-ribosylated by CT unless G beta was added back. RM/1 antiserum against Gs alpha was used to immunoprecipitate Gs, and the subunit composition of the immunoprecipitate was determined. When Gs was incubated with 2 mM MgCl2, the Gs heterotrimer was immunoprecipitated, and Gs alpha could be ADP-ribosylated by CT. Activation of Gs with GTP gamma S or fluoroaluminate in the presence of 2 mM MgCl2 did not cause Gs subunit dissociation nor did it affect the ability of Gs alpha to be ADP-ribosylated by CT. MgCl2 caused a dose-dependent decrease in the amount of G beta that coprecipitated with Gs alpha in the absence as well as the presence of GTP gamma S or fluoroaluminate. Gs subunit dissociation was accompanied by a corresponding decrease in CT-catalyzed ADP-ribosylation of Gs alpha regardless of whether or not GTP gamma S or fluoroaluminate was bound to Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

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Fluoroaluminate had no effect on cAMP levels in cells but inhibited agonist-stimulated cAMP accumulation. In membranes, fluoroaluminate stimulated adenylate cyclase activity between 1 and 10 mM but not at higher concentrations, and it inhibited agonist-stimulated adenylate cyclase activity at concentrations greater than 1 mM. Fluoroaluminate is known to activate Gs and Gi, the guanine nucleotide-binding (G) proteins that stimulate and inhibit adenylate cyclase. G proteins are heterotrimeric, with unique alpha and common beta gamma subunits, and activation involves dissociation of alpha from beta gamma. Pertussis toxin catalyzes ADP-ribosylation of alpha i of heterotrimeric Gi but not free alpha i. Fluoroaluminate prevented pertussis toxin-catalyzed ADP-ribosylation of Gi in cells and membranes, suggesting that Gi is activated by fluoroaluminate in both. Cholera toxin catalyzes ADP-ribosylation of the alpha s subunit of Gs. In cells, agonist often increased cholera toxin-catalyzed ADP-ribosylation of Gs, but fluoroaluminate decreased ADP-ribosylation even in the presence of agonist, suggesting that Gs cannot be activated in the presence of fluoroaluminate. In membranes, both agonist and fluoroaluminate increased cholera toxin-catalyzed ADP-ribosylation, suggesting that Gs is activated by these agents. We conclude that fluoroaluminate activates Gi but not Gs in cells and activates both G proteins in membranes. The value of bacterial toxins in assessing the state of G protein in cells and membranes is demonstrated.

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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.

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A single compound with sex pheromone activity was isolated from the female soybean cyst nematode,Heterodera glycines, by a sequence of four high-performance liquid chromatographic steps and identified as vanillic acid by a combination of ultraviolet spectroscopy and chromatography. The structure was confirmed by gas chromatography-mass spectrometry. Both attractancy and coiling behavior in male soybean cyst nematode were elicited by authentic vanillic acid.

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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)

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The murine Leydig tumor cell line, MLTC-1, has a gonadotropin-responsive adenylate cyclase system. Binding of human chorionic gonadotropin (hCG) stimulates the accumulation of cyclic AMP in these cells. Chemically deglycosylated hCG (DG-hCG) is an antagonist that binds with high affinity to the gonadotropin receptor, but fails to stimulate adenylate cyclase. This antagonism can be reversed if the binding of DG-hCG is followed by treatment of the DG-hCG-receptor complex with antibodies against hCG. Polyclonal antibodies against DG-hCG were raised in rabbits. These antibodies were strongly cross-reactive with hCG, bound to both the alpha- and beta-subunits of hCG and DG-hCG, and reversed the antagonism of DG-hCG. The antiserum was divided into two fractions by affinity chromatography on hCG-Sepharose. The fraction that was not retained reacted only with DG-hCG (DG-hCG antibodies) and, on Western blots, bound to both the alpha- and beta-subunits of DG-hCG. DG-hCG antibodies did not reverse the antagonism of DG-hCG. However, using 125I-protein A, we were able to detect binding of these antibodies to the cell surface DG-hCG-receptor complex. The fraction of antibodies retained by the affinity column reacted with both DG-hCG and hCG (DG-hCG/hCG antibodies). On Western blots, DG-hCG/hCG antibodies bound to the beta-subunit, but only weakly to the alpha-subunit of both hCG and DG-hCG. These antibodies also bound to the cell surface DG-hCG-receptor complex. In addition, DG-hCG/hCG antibodies were able reverse the antagonism of DG-hCG. Reversal of DG-hCG antagonism by the whole antiserum was blocked by the beta- but not the alpha-subunit of hCG. Polyclonal antiserum against the beta- but not the alpha-subunit of hCG reversed the antagonism of DG-hCG. From these results, we conclude that antibody binding to specific determinants common to both native and deglycosylated beta-subunit reverses the antagonism of DG-hCG. In addition, antibodies directed against unique determinants on the deglycosylated beta-subunit are not capable of reversing the antagonism of DG-hCG.

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Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.

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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.

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Population dynamics, rate of root penetration, and external root feeding behavior of Pratylenchus agilis (Pa) in monoxenic cultures of intact corn seedlings and root explants of corn, tomato, and soybean were studied. In descending order of suitability as hosts were I. O. Chief corn, Rutgers tomato, and Williams soybean. Soybean entries Kent, Pickett 71, PI 90763, and Essex were poor hosts. Numbers of eggs and vermiform Pa in the agar medium indicated total fecundity and host suitability. Agar, sand, or soil as support media did not appear to affect Pa root penetration, but the rate of corn root growth did. Whereas most vermiform Pa and eggs were in roots, substantial numbers appeared able to feed and complete their life cycle as ectoparasites on root epidermal cells and root hairs.

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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.

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Loss of gonadotropin receptors in murine Leydig tumor cells and of beta-adrenergic receptors in rat glioma C6 cells occurred following exposure of the cells to human chorionic gonadotropin and isoproterenol, respectively. Down-regulation of receptors was mimicked in part by other agents that elevated cyclic AMP levels in the cells such as cholera toxin and dibutyryl cyclic AMP. Whereas agonist-mediated receptor loss was rapid and almost total, down-regulation by cyclic AMP was slower and less extensive. Down-regulation of receptors did not appear to be accompanied by loss of the regulatory and catalytic components of adenylate cyclase. Hormone-mediated down-regulation was preceded by desensitization of hormone-stimulated adenylate cyclase. In contrast, there was no evidence that cyclic AMP caused desensitization. Finally, loss of receptors induced either by agonists or cyclic AMP required protein synthesis as cycloheximide inhibited down-regulation. We conclude that down-regulation of receptors in these cells is a complex process involving both cyclic AMP-independent and -dependent events.

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