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Scavenger receptor class B type 1 (SR-B1), an HDL receptor plays a crucial role in cholesterol metabolism in the liver, steroidogenic tissues, and vascular cells including macrophages. SR-B1 is subject to regulation at the transcription, posttranscription and posttranslational levels. We previously provided evidence that PDZ domain containing NHERF1 and NHERF2 regulate SR-B1 protein levels post-transcriptionally, although the underlying mechanism(s) by which NHERF1 and NHERF2 regulate SR-B1 protein levels is not well understood. In this study, we demonstrate that SR-B1 is degraded intracellularly via ubiquitin-proteasome pathway and that SR-B1 can be ubiquitinated at K500 and K508 residues. Overexpression of NHERF1 or NHERF2 enhanced SR-B1 ubiquitination and degradation. NHERF1 and NHERF2 promote SR-B1 ubiquitination at sites K508 and K500, respectively. These results suggest that NHERF1 and NHERF2 down-regulated SR-B1 at least in part via the ubiquitin/proteasome pathway.

Rat adrenocortical cells in culture respond to stimulation by ACTH alone (15 fold over basal) and to ACTH + added lipoproteins (as an exogeneous source of cholesterol), with an additional 25–30 fold rise in steroidogenesis. With the addition of okadaic acid (OKA, 100 nM), a potent protein phosphatase inhibitor, the lipoprotein-induced rise in steroidogenesis is blocked. If 20 α-hydroxycholesterol is provided instead of lipoprotein-cholesterol, OKA has no effect suggesting that OKA affects only actively transported cholesterol. Since the OKA block is preceded by specific morphological changes in the cell (i.e., the loss of Golgi-associated microtubules followed by the disruption of the Golgi apparatus itself), it is hypothesized that some OKA-sensitive phosphoprotein associated with the microtubule/Golgi network of adrenocortical cells is critical for lipoprotein-derived cholesterol uptake and/or transport during steroidogenesis.

We have examined the protein kinase C that are present in mouse, rat, guinea pig and rabbit liver. Initial subcellular fractionation analysis indicated that the majority (75–85%) of the activity was associated with particulate fraction of the liver. The bound protein kinase C was dissociated by homogenization of livers in buffer containing EGTA, EDTA and various proteolytic inhibitors and the solubilized extract was used to resolve multiple forms of the enzyme. The fractionation procedure, sequentially utilized (NH 4 ) 2 SO 4 precipitation, ion exchange chromatography, gel permeation chromatography, and hydroxylapatite column chromatography. With hydroxylapatite, protein kinase C was resolved into three isoenzymic forms designated C-I, C-II and C-III. In each case, the predominant activity consisted of C-II and C-III and together they represented about 80–88% of the total activity. All three isoenzymes from each source demonstrated an absolute requirement for PS + Ca 2+ (∼25-50 fold stimulation over basal activity); for maximal activity the isoenzymes also required the presence of divalent metal ion, Mg 2+ (5–10 mM) and lysine rich histone (H 1 ). Both diolein and TPA decreased the Ca 2+ and PS requirement of the enzyme and directly stimulated enzyme activity in the presence of suboptimal concentrations of Ca 2+ and PS. In conclusion, the present studies suggest that protein kinase C in mammalian liver exists in isoenzymic forms.

SUMMARY: Rat ovarian cells were exposed to cholera enterotoxin, and the effect on progesterone synthesis as well as on protein kinase stimulation was examined. Cholera enterotoxin stimulated ovarian steroidogenesis in a dose dependent manner similar to that of hCG. The stimulation of protein kinase by cholera toxin was followed by a lag period, whereas hCG effect was imnediate. Mixed gangliosides, when added to the incubation medium, blocked the cholera enterotoxin-stimulated protein kinase activity and abolished the decrease in exogenous [sH] cyclic AMP receptor activity brought about by the toxin. In contrast, under similar experimental conditions ganglioside addition elicited no effect on protein kinase activation produced by hCG or LH. The data suggest that gangliosides do not appear to be directly involved in gonadotropin binding to ovarian cell membrane and subsequent mediation of physiological response.

fifty-fold increase in progesterone production. Both cholera enterotoxin and hCG-stimulated progesterone response was accompanied by a lag period. The duration of the lag period in the production of the progesterone depended on the concentration ot gonadotropin or cholera enterotoxin, and with maximally stimulating dose it was 20-30.minutes. Addition of highly purified mixed gangliosides to the incubation medium abolished the stimulatory effect of cholera enterotoxin on progesterone response. In contrast, under iaentical experimental conditions, ganglioside addition produced no effect on progesterone response elicited by hCG or LH. Similarly mixed gangliosides did not prevent the specific binding of [ 1251]hCG to the ovarian cells or to the membranes isolated from the ovary. In addition preincubation of [12!jI]hCG with ganglioside did not alter the subsequent binding of the hormone to the ovarian cell surface receptor. These findings suggest that gangliosides are not involved in the hormone receptor interactions and subsequent receptor mediated physiological response.

In rat anterior pituitary cell cultures GnRH (1nM) stimulated a progressive increase in LH release into the medium from 1 to 8 h of incubation, while cellular LH showed a corresponding decrease. GnRH (1nM) neither modified the uptake nor the incorporation of [3H]-glucosamine and [3H]-proline into total protein. The incorporation of [3H]-proline into cellular LH also was unaffected by GnRH. In contrast, GnRH stimulated a 3 to 4-fold increase in [3H]-glucosamine incorporation into cellular LH. The agonistic analogue, [des GlyNH210]-LHRH ethylamide, mimicked the GnRH effects and was 5 to 6 times more potent than GnRH. The antagonistic analogue, [D-Phe2, D-Phe6]-LHRH blocked the GnRH-stimulated effects. These results suggest that GnRH and agonistic analogues may preferentially regulate turnover or synthesis of the carbohydrate moiety of LH.