G protein ß4 as a structural determinant of enhanced nucleotide exchange in the A2AAR-Gs complex.

Adenosine A2A receptors (A2AAR) evoke pleiotropic intracellular signaling events via activation of the stimulatory heterotrimeric G protein, Gs. Here, we used cryoEM to solve the agonist-bound structure of A2AAR in a complex with full-length Gs α and Gß4γ2 (A2AAR-Gs α:ß4γ2). The orthosteric binding site of A2AAR-Gs α:ß4γ2 was similar to other structures of agonist-bound A2AAR, with or without Gs. Unexpectedly, the solvent accessible surface area within the interior of the complex was substantially larger for the complex with Gß4 versus the closest analog, A2AAR-miniGs α:ß1γ2. Consequently, there are fewer interactions between the switch II in Gs α and the Gß4 torus. In reconstitution experiments Gß4γ2 displayed a ten-fold higher efficiency over Gß1γ2 in catalyzing A2AAR dependent GTPγS binding to Gs α. We propose that the less constrained switch II in A2AAR-Gs α:ß4γ2 accounts for this increased efficiency. These results suggest that Gß4 functions as a positive allosteric enhancer versus Gß1.

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry.

The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, "crossmember" helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity.

ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels.

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.

A quantized mechanism for activation of pannexin channels.

Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels.

The second extracellular loop of the adenosine A1 receptor mediates activity of allosteric enhancers.

Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.

Binding of ß4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry.

Characterization of G protein ßγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native ßγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and ßγ labeled with heavy isotopes purified. Heavy ßγ was combined with either recombinant ßγ purified from Sf9 cells, ßγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched ßγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing ß and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three ß isoforms, ß(1), ß(2), and ß(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. ß(1) and γ(5) were most abundant in the enriched ßγ fraction, and this ßγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more ß(4) and γ(5) compared to the enriched ßγ fraction; also, more ß(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched ßγ fraction. These results suggest that preferences for particular ßγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.

Progesterone receptor repression of prolactin/signal transducer and activator of transcription 5-mediated transcription of the beta-casein gene in mammary epithelial cells.

Prolactin (PRL) and glucocorticoids act synergistically to stimulate transcription of the beta-casein milk protein gene. Signal transducer and activator of transcription 5 (Stat5) mediates PRL-dependent trans-activation, and glucocorticoid potentiation occurs through cross talk between glucocorticoid receptor (GR) and Stat5 at the beta-casein promoter. In the mouse, progesterone withdrawal leads to terminal differentiation and secretory activation of the mammary gland at parturition, indicating progesterone's role in repressing milk protein gene expression during pregnancy. To investigate the mechanism of the inhibitory action of progesterone, experiments were performed with cell culture systems reconstituted to express progesterone receptor (PR), the PRL receptor/Stat5 signaling pathway, and GR, enabling evaluation of PR, GR, and Stat5 interactions at the beta-casein promoter. With COS-1, normal murine mammary gland, HC-11, and primary mammary epithelial cells, progestin-PR directly repressed the PRL receptor/Stat5a signaling pathway's mediation of PRL-induced beta-casein transcription. Progestin-PR also inhibited glucocorticoid-GR enhancement of PRL induced trans-activation of beta-casein. Inhibition depended on a functional PR DNA binding domain and specific PR-DNA interactions at the beta-casein promoter. Chromatin immunoprecipitation assays in HC-11 cells revealed recruitment of PR and Stat5a to the beta-casein promoter by progestin or PRL, respectively. Recruitment was disrupted by cotreatment with progestin and PRL, suggesting a mutual interference between activated PR and Stat5a. Without PRL, progestin-PR also recruited Stat5a to the beta-casein promoter, suggesting that recruitment of an unactivated form of Stat5a may contribute to inhibition of beta-casein by progesterone. These results define a negative cross talk between PR and Stat5a/GR that may contribute to the physiological role of progesterone to repress lactogenic hormone induction of the beta-casein gene in the mammary gland during pregnancy.

Progesterone receptor transcription and non-transcription signaling mechanisms.

The diverse effects of progesterone on female reproductive tissues are mediated by the progesterone receptor (PR), a member of the nuclear receptor family of ligand-dependent transcription factors. Thus, PR is an important therapeutic target in female reproduction and in certain endocrine dependent cancers. This paper reviews our understanding of the mechanism of action of the most widely used PR antagonist RU486. Although RU486 is a competitive steroidal antagonist that can displace the natural hormone for PR, it's potency derives from additional "active antagonism" that involves inhibiting the activity of PR hormone agonist complexes in trans through heterodimerization and competition for binding to progesterone response elements on target DNA, and by recruitment of corepressors that have the potential to actively repress gene transcription. An additional functional role for PR has recently been defined whereby a subpopulation of PR in the cytoplasm or cell membrane is capable of mediating rapid progesterone induced activation of certain signal transduction pathways in the absence of gene transcription. This paper also reviews recent results on the mechanism of the extra-nuclear action of PR and the potential biological roles and implications of this novel PR signaling pathway.

Mechanism of action of progesterone antagonists.

The effects of progesterone on target tissues are mediated by progesterone receptors (PRs), which belong to a family of nuclear receptors and function as ligand-activated transcription factors to regulate the expression of specific sets of target genes. Progesterone antagonists repress the biological actions of progesterone by "actively" inhibiting PR activation. This work discusses the first clinically used progesterone antagonist RU486 and closely related compounds in terms of how these compounds inhibit progesterone action through heterodimerization and competition for DNA binding and by the recruitment of corepressors to promoters of target genes to repress transcription. We discuss cellular factors that may influence the activity of these compounds, such as the availability of coactivators and corepressors and the context of specific target promoters in any given cell type. We also discuss steroidal and nonsteroidal antagonist selectivity for PR versus other steroid hormone receptors and suggest that it may be possible to develop tissue/cell specific modulators of PR.