Adenylyl Cyclase Activity In Cells Research Articles

Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors α and β in transfected cells

Recently, a unique family of membrane progestin receptors (mPRalpha, mPRbeta, and mPRgamma) was identified, which may be responsible for mediating rapid, nongenomic actions of progestins in a variety of target tissues. In this study, the mPRalpha and mPRbeta isoforms from zebrafish were shown to be rapidly and specifically activated by the maturation-inducing steroid (MIS) of this species, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). The zebrafish mPRalpha and a previously uncharacterized mPRbeta isoform were stably expressed in nuclear progesterone receptor-deficient mammalian breast cancer cells, MDA-MB-231. Expression and surface localization of the receptors were verified by flow cytometry, biotin surface labeling, and Western blotting. Plasma membrane proteins from mPRalpha- or mPRbeta-transfected cells showed high affinity (mPRalpha, K(d) 7 nM; mPRbeta, K(d) 12 nM), saturable, displaceable, single-binding sites specific for 17,20beta-DHP, whereas negligible specific 17,20beta-DHP binding was observed in nontransfected cells. Progestin treatment caused significant activation of mitogen-activated protein kinase (MAPK) within 5 min in cells transfected with either of the receptors as measured by western blotting and flow cytometry. The rank order of the potencies of several progestins in activating MAPK via mPRalpha and mPRbeta was the same (17,20beta-DHP>progesterone >4-pregnen-17,20beta,21-triol-3-one). Interestingly, the MIS in zebrafish, 17,20beta-DHP, was also the most potent inhibitor, among the progestins tested, of adenylyl cyclase activity in cells transfected with either of the receptors. This progestin significantly decreased cAMP levels in both mPRalpha- and mPRbeta-transfected cells in a dose-responsive and time-dependent manner. In addition, signaling of the zebrafish mPRalpha was blocked by pertussis toxin, implying activation of a G(i) protein, while sensitivity to pertussis or cholera toxin was not shown with mPRbeta-mediated signaling, possibly indicating that this receptor activates a different pertussis toxin-insensitive G protein. The results of this study suggest that zebrafish mPRalpha and mPRbeta signal similarly upon progestin binding resulting in rapid activation of MAPK and downregulation of adenylyl cyclase activity.

A Role for the Distal Carboxyl Tails in Generating the Novel Pharmacology and G Protein Activation Profile of μ and δ Opioid Receptor Hetero-oligomers

Opioid receptor pharmacology in vivo has predicted a greater number of receptor subtypes than explained by the profiles of the three cloned opioid receptors, and the functional dependence of the receptors on each other shown in gene-deleted animal models remains unexplained. One mechanism for such findings is the generation of novel signaling complexes by receptor hetero-oligomerization, which we previously showed results in significantly different pharmacology for mu and delta receptor hetero-oligomers compared with the individual receptors. In the present study, we show that deltorphin-II is a fully functional agonist of the mu-delta heteromer, which induced desensitization and inhibited adenylyl cyclase through a pertussis toxin-insensitive G protein. Activation of the mu-delta receptor heteromer resulted in preferential activation of Galpha(z), illustrated by incorporation of GTPgamma(35)S, whereas activation of the individually expressed mu and delta receptors preferentially activated Galpha(i). The unique pharmacology of the mu-delta heteromer was dependent on the reciprocal involvement of the distal carboxyl tails of both receptors, so that truncation of the distal mu receptor carboxyl tail modified the delta-selective ligand-binding pocket, and truncation of the delta receptor distal carboxyl tail modified the mu-selective binding pocket. The distal carboxyl tails of both receptors also had a significant role in receptor interaction, as evidenced by the reduced ability to co-immunoprecipitate when the carboxyl tails were truncated. The interaction between mu and delta receptors occurred constitutively when the receptors were co-expressed, but did not occur when receptor expression was temporally separated, indicating that the hetero-oligomers were generated by a co-translational mechanism.

Differential chemokine activation of CC chemokine receptor 1‐regulated pathways: ligand selective activation of Gα 14‐coupled pathways

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptor-ligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1-coupled pathways by known CCR1 agonists. Chemokines leukotactin-1, macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1delta all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1delta was unable to signal via G14-, G16- or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and Galpha14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G(i/o)-coupled pathways, but MIP-1delta, HCC-1 and MPIF-1 were unable to activate G14-mediated stimulation of phospholipase Cbeta activity. In addition, MIP-1delta was unable to promote the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. This suggests that different chemokines are able to selectively activate CCR1-coupled pathways, probably because of different intrinsic ligand efficacies. CCR1 and Galpha14 or Galpha16 are co-expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine-tune intracellular signaling pathways.

Pharmacological Survey of Medicinal Plants for Activity at Dopamine Receptor Subtypes. II. Screen for Binding Activity at the D1 and D2 Dopamine Receptor Subtypes

Aqueous, organic and alcoholic extracts of 163 Bolivian, Chinese and Pakistani medicinal plants were evaluated for their ability to inhibit the binding of radioligands selective for the D1 and D2 dopamine receptor subtypes expressed in Sf9 cells. Based upon the results of the initial screen, 25 extracts were selected for further evaluation. Concentrationresponse competitive radioligand binding studies were performed to obtain IC 50 values for the inhibition of the binding at D1 or D2 receptors by the selected extracts. The D1:D2 receptor ratio of IC 50 values for the extracts ranged from approximately two-fold selective for the D1 receptor to threefold selective for the D2 receptor. D1 and D2 dopamine receptors are known to be linked to the activation and inhibition of adenylyl cyclase activity, respectively. Therefore, the selected extracts were tested for the ability to either a) stimulate adenylyl cyclase activity using stably transfected HEK 293 cells expressing human D1 receptors (hD1-HEK) or b) inhibit forskolin-dependent stimulation of adenylyl cyclase activity in stably transfected HEK 293 cells expressing rat D2 dopamine receptors (rD2-HEK). Two extracts were found to stimulate adenylyl cyclase activity in hD1-HEK cells. However, similar results were observed when untransfected HEK cells were used, suggesting that the ability of these two extracts to increase cAMP levels in hD1-HEK cells was not mediated through a D1-like dopamine receptor. While the majority of extracts did not have an effect on forskolin-dependent stimulation in rD2-HEK cells, six extracts were capable of inhibiting adenylyl cyclase activity in these cells. For five of these extracts, the inhibitory effect appeared to be dependent upon the expression of D2 receptors because no inhibitory effect was observed when untransfected HEK 293 cells were used. In summary, the results of this preliminary radioligand binding and functional activity screen of extracts from medicinal plants for pharmacologic activity at D1 and D2 dopamine receptors indicated that the majority of components capable of binding to the D1 and D2 dopamine receptor subtypes appeared to be essentially nonselective. While none of the selected extracts were found to have agonist activity at D1 receptors, several of the extracts exhibited inverse agonist activity at D1 dopamine receptors. Five of the extracts appeared to be agonists at D2 dopamine receptors. Therefore, several classes of extracts were identified which appeared to contained unique combinations of the following pharmacologic properties: D1 dopamine receptor antagonist, D2 dopamine receptor antagonist, D1 receptor inverse agonists and D2 receptor agonists. In addition, two of the extracts tested appeared to modulate adenylyl cyclase activity, but this activity did not appear to be mediated through activation of a dopamine receptor subtype.

Chemokine receptor CCR5 functionally couples to inhibitory G proteins and undergoes desensitization.

Chemokine receptor CCR5 is not only essential for chemotaxis of leukocytes but also has been shown to be a key coreceptor for HIV-1 infection. In the present study, hemagglutinin epitope-tagged human CCR5 receptor was stably expressed in Chinese hamster ovary cells or transiently expressed in NG108-15 cells to investigate CCR5-mediated signaling events. The surface expression of CCR5 was confirmed by flow cytometry analysis. The CCR5 agonist RANTES stimulated [35S]GTPgammaS binding to the cell membranes and induced inhibition on adenylyl cyclase activity in cells expressing CCR5. The effects of RANTES were CCR5 dependent and could be blocked by pertussis toxin. Furthermore, overexpression of Gialpha2 strongly increased both RANTES-dependent G-protein activation and inhibition on adenylyl cyclase in cells cotransfected with CCR5. These data demonstrated directly that activation of CCR5 stimulated membrane-associated inhibitory G proteins and indicated that CCR5 could functionally couple to G-protein subtype Gialpha2. The abilities of CCR5 to activate G protein and to inhibit cellular cAMP accumulation were significantly diminished after a brief prechallenge with RANTES, showing rapid desensitization of the receptor-mediated responsiveness. Prolonged exposure of the cells to RANTES caused significant reduction of surface CCR5 as measured by flow cytometry, indicative of agonist-dependent receptor internalization. Our data thus demonstrated that CCR5 functionally couples to membrane-associated inhibitory G proteins and undergoes agonist-dependent desensitization and internalization.

Pretreatment with 17β-Estradiol Attenuates Basal- and PTH-Stimulated Membrane Adenylyl Cyclase Activity in Human Osteoblast-like SAOS-2 Cells

Parathyroid hormone (PTH)-stimulated production of cAMP by intact human osteoblast-like SaOS-2 cells is diminished by pretreatment with 17β-estradiol (E2). The goal of the present study was to determine whether E2affected adenylyl cyclase activity in cell membranes. Cells were deprived of steroid hormones for 24 h and then incubated with E2(1 nM) or vehicle for 12 h. Cell membranes were prepared and incubated with [α-32P]-ATP in the absence or presence of agonist, and the amount of [32P]-cAMP produced was measured to quantify adenylyl cyclase activity. There was less cAMP produced by membranes from E2-treated cells in response to PTH, GTPγS, forskolin, and Mn2+. E2had no effect on the amounts of Gsor Gi. We propose that pretreatment of SaOS-2 cells with E2reduced membrane cyclase activity, at least in part, by actions on the PTH-sensitive adenylyl cyclase and/or the G protein-adenylyl cyclase complex.

Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor.

A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP-binding protein (G protein)-coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ, has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.