Heterotrimetric Gi/o proteins control cyclic AMP oscillations and cytoskeletal structure assembly in primary human granulosa-lutein cells.

In granulosa cells, the luteinising hormone (LH) and the follicle-stimulating hormone (FSH) receptors are coupled to the adenylyl cyclase-cAMP pathway. We identified at least eight different G proteins belonging to three families--Gs, Gq, and Gi/o--in primary human granulosa-lutein cells. By exploring the function of Gi/o by time-lapse and digital-imaging microscopy of live cells, we found that the reversible actin stress fibre-dependent cytoplasmic retraction of pre-luteinised cells in primary culture is a highly sensitive and quite rapid system allowing detection of an intracellular cAMP surge. This morphology was characterised by maintenance of connexin43-dependent cell-cell contacts and that of microtubule-directed cell processes attached to the substrate and to neighbouring cells. Inhibitors of cyclic nucleotide phosphodiesterase subfamily type 4 (PDE-4), hLH and hFSH provoked this reversible cAMP-dependent phenotype in a temporal-, spatial- and dose-dependent manner. Gi/o inhibited adenylyl cyclase in membranes, and cell treatment with islet-activating protein (IAP) caused the cAMP-dependent retracted phenotype. It is concluded that the basal intracellular cAMP level is kept within a narrow range of concentrations, below the threshold for disassembly of stress fibres, through Gs, Gi/o, adenylyl cyclases and phosphodiesterase-4. This work supports the paradigm that switching of the agonist-occupied receptors to Gs and Gi/o would control both the intracellular bursts of cAMP (through the gonadotropin-catalysed activation of Gs) and the basal cAMP (through a Gi/o-mediated braking effect).

Rho proteins are localized with different membrane compartments involved in vesicular trafficking in anterior pituitary cells.

In order to explore the role of certain GTP binding proteins in the rat anterior pituitary, we have analyzed the subcellular distribution of the proteins rho and rab. They were found in both membrane and cytosolic fractions. Rab1 and rab2 were localized in both Golgi and endoplasmic reticulum (ER) membranes, while rab4 and rab6 were found in fractions enriched with Golgi and plasma membranes, implicating these proteins in the control of vesicular intracellular trafficking as described in other systems. Rab3 was localized like a fraction of synaptophysin, suggesting a role for rab3 in the targeting of "synaptic-like' microvesicles. We have identified three substrates of C. botulinum exoenzyme C3. A 26-kDa substrate with an isoelectric point (pI) of 5.2, probably rhoB, was localized in the lightest fractions such as rab3 and synaptophysin proteins. Two other 23-24 kDa substrates with pI of 5.5-5.8, probably rhoA and/or rhoC, were found in both fractions enriched with ER and secretory granules. Rho proteins have been implicated in the control of actin polymerization. Their localization in anterior pituitary suggests that rhoB could control the association of synaptic-like microvesicles and plasma membrane, and that rhoA/rhoC could play a role in secretory granule exocytosis; these two pathways being involved in cytoskeleton protein reorganisation in response to extracellular signals.

ADP-ribosylation of G alpha i and G alpha o in pituitary cells enhances their recognition by antibodies directed against their carboxyl termini.

Using antibodies raised against synthetic peptides of heterotrimeric GTP binding proteins, we demonstrate the presence of G alpha s, G alpha i1,2, G alpha i3, G alpha o2, and G beta subunits in pituitary cells. Pretreatment of pituitary cells with cholera toxin diminished the immunoreactivity of G alpha s and this decrease was kinetically coupled to the rate of G alpha s ADP-ribosylation. ADP-ribosylation by islet activating protein (IAP or Bordetella pertussis toxin) of G alpha i and G alpha o enhanced their immunoreactivities to antibodies raised against synthetic decapeptides that correspond to the G alpha carboxyl termini. Such enhancement was not observed when antibodies directed against the NH2-termini were used. These findings are consistent with the fact that ADP-ribosylation by IAP occurs on the cysteine located in the carboxyl terminal part of G alpha i and G alpha o. These observations mean that the kinetics and extent of Gi and Go ADP-ribosylation by IAP in whole pituitary cells and membrane preparations can be followed. It could be that ADP-ribosylation causes conformational changes in G alpha i and G alpha o. Indeed, we observed that ADP-ribosylated G alpha i was more sensitive to trypsin proteolysis and that the ADP-ribosylation rates of G alpha i and G alpha o in whole cells were comparable to the rate of loss of coupling between inhibitory neurohormone receptors and adenylyl cyclase.

Vip-induced cross-talk between G-proteins in membranes from rat anterior pituitary cells.

In order to study the activation mechanism of heterotrimeric G-proteins by agonist-liganded receptors, GTP gamma S binding to membranes was measured in rat adenohypophyseal cells after addition of dopamine (DA) or vasoactive intestinal peptide (VIP), which, respectively, inhibit and activate pituitary adenylyl cyclase. G-protein subunit present in anterior pituitary cells was characterized by either ADP-ribosylation catalysed by Bordetella pertussis and cholera toxins or by immunoblot using specific antisera. Binding of GTP gamma S was found to depend upon GTP gamma S and Mg2+ concentrations; it was sensitive to pretreatment of the cells with cholera and Bordetella pertussis toxins (IAP). DA increased binding of the nucleotide. Paradoxically, VIP decreased the rate of GTP gamma S binding; the effect was suppressed by prior treatment of the cells with either cholera toxin or IAP. VIP also increased [33P]ADPribose incorporation in Gi/Go-proteins catalysed by IAP. Forskolin was also able to decrease GTP gamma S binding, thus suggesting that the binding of forskolin with the adenylyl cyclase catalytic unit might activate Gs proteins through an increased interaction between Gs and adenylyl cyclase. Taken together, these results suggest that VIP, as well as forskolin, may both accelerate the activation of Gs and suppress the inhibitory effect of activated Gi/Go-proteins. Interactions between Gs and Gi/Go subunits mediated by beta gamma and/or adenylyl cyclase might thus result in a kinetic coupling of transduction pathways involving distinct G-proteins.

Adenylate cyclase stimulation and luteinizing hormone-receptor interaction in plasma membranes from rat testicular interstitial cells in relation to the chemical structure of the hormone. Role of Mg2+.

To understand more closely the structural requirements of the LH molecule necessary to stimulate adenylate cyclase, we studied the modulation of this enzyme in partially purified plasma membranes prepared from isolated interstitial cells of rat testis submitted to oLH and to some oLH derivatives and natural analogues. The role of Mg2+ was also investigated in relation to the structural modifications of oLH. Some new facts appeared in this study: 1. Methyl oLH, which exhibited the same ability as native oLH to stimulate cAMP accumulation and steroidogenesis in isolated cells, cannot induce the same level of maximal stimulation of adenylate cyclase as native oLH in plasma membranes. This phenomenon is related to the Mg2+ concentration, and the differences between maximal activation induced by methyl oLH and oLH were more apparent at a free Mg2+ concentration of 3.3 mmol/l than at lower concentrations. The maximal activity (in terms of native oLH) of other alkyl derivatives, such as ethyl or isopropyl oLH, on the contrary, was similar in isolated plasma membranes and in intact cells suggesting that the differential behaviour of the membranes specifically concerns the methyl derivative. 2. Guanidyl oLH and guanidyl porcine LH, which were able to induce cAMP accumulation in intact cells, did not exhibit any stimulating activity in plasma membranes. 3. Among the natural analogues, hCG and pLH are distinguished by a lower maximal activity (by comparison with oLH) particularly at high Mg2+ concentration. This work shows that changes in the LH structure have an impact not only on the parameters of the adenylate cyclase complex but also on the transduction of the hormone signal and its modulation by Mg2+.

Modulation of adenylate cyclase by guanine nucleotides and Kirsten sarcoma virus mediated transformation.

Certain tumour cells contain activated ras genes that code for 21 000 dalton proteins (p21). These proteins associate with the inner face of the plasma membrane and bind guanine nucleotides specifically. In order to determine whether p21s have functions similar to other GTP binding proteins, we investigated the regulation, by guanine nucleotides, of adenylate cyclase (AC) activity in membrane preparations isolated from fibroblasts (C127) transformed by a temperature sensitive mutant of Kirsten sarcoma virus (Ts 371). The degree of AC stimulation by GMP P(NH)P increased when these cells were shifted from the permissive temperature (33 degrees C) to the non-permissive temperature (39 degrees C). This effect was more pronounced at low Mg++ and low GMP P(NH)P concentrations. AC stimulation remained unchanged in rat fibroblasts infected with a temperature sensitive mutant of Rous Sarcoma virus. AC activity was depressed in C127 cells infected with wild type KiMSV. Our data illustrate the feasibility of correlating alterations in the AC system with ras gene expression and using such experimental approaches to elucidate the physiological functions of the p21 proteins.

Effects of antimicrotubular agents in cAMP production and in steroidogenic response of isolated rat Leydig cells.

In dispersed rat Leydig cells, colchicine was found to stimulate basal cAMP production and testosterone secretion in a dose and time-dependent manner, but to a lesser extent than LH. However, these drugs are unable to stimulate adenylate cyclase activity in plasma membranes isolated from these cells. The amount of testosterone secreted at 150 min under the influence of colchicine and LH added simultaneously was not different from the amount produced during stimulation by LH alone. It is only after exposure of the cells for 1 hr to colchicine that the accumulation of cAMP in response to LH was inhibited; furthermore, both intracellular and medium testosterone accumulation in response to the hormone were reduced. Similar effects were observed with two other alkaloids, vinblastine and podophyllotoxin. The three drugs also inhibited the stimulation of testosterone secretion by 8-Br-cAMP or choleratoxin. These studies suggest that the state of microtubule polymerization and/or tubulin can influence the process of steroidogenesis in rat Leydig cells.

Inhibin-like activity and steroid content in human follicular fluid during the periovulatory period.

Inhibin-like activity as well as estradiol-17 beta and progesterone content were assayed in human follicular fluid obtained at different stages of the periovulatory period. Between the start of the midcycle estrogen surge in plasma and the subsequent gonadotrophin peak, follicular fluid was found to contain similar levels of estradiol-17 beta and progesterone (8.2 pmol/microliter) and to induce a marked (44%) inhibition of gonadotrophin-releasing hormone-stimulated release of FSH. The midcycle gonadotrophin peak was characterized by a drop in estradiol-17 beta levels and a tripling in progesterone levels, whereas inhibin-like activity fell to almost half the previous value.