Nathan E. Hall

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand.

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M2 mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M2 mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[3H]methylscopolamine, but in functional assays of M2 mAChR-mediated ERK1/2 phosphorylation and guanosine 5′-3-O-([35S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M2 mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of “directed efficacy” whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.

Identification of Orthosteric and Allosteric Site Mutations in M2 Muscarinic Acetylcholine Receptors That Contribute to Ligand-selective Signaling Bias

Muscarinic acetylcholine receptors contain at least one allosteric site that is topographically distinct from the acetylcholine, orthosteric binding site. Although studies have investigated the basis of allosteric modulation at these receptors, less is known about putative allosteric ligands that activate the receptor in their own right. We generated M2 muscarinic acetylcholine receptor mutations in either the orthosteric site in transmembrane helices 3 and 6 (TM3 and -6) or part of an allosteric site involving the top of TM2, the second extracellular (E2) loop, and the top of TM7 and investigated their effects on the binding and function of the novel selective (putative allosteric) agonists (AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl), 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)-3,3-dihydro-2(1H)-quinolinone), and N-desmethylclozapine) as well as the bitopic orthosteric/allosteric ligand, McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium). Four classes of agonists were identified, depending on their response to the mutations, suggesting multiple, distinct modes of agonist-receptor interaction. Interestingly, with the exception of 77-LH-28-1, allosteric site mutations had no effect on the affinity of any of the agonists tested, but some mutations in the E2 loop influenced the efficacy of both orthosteric and novel selective agonists, highlighting a role for this region of the receptor in modulating activation status. Two point mutations (Y1043.33A (Ballesteros and Weinstein numbers in superscript) in the orthosteric and Y177A in the allosteric site) unmasked ligand-selective and signaling pathway-selective effects, providing evidence for the existence of pathway-specific receptor conformations. Molecular modeling of 77-LH-28-1 and N-desmethylclozapine yielded novel binding poses consistent with the possibility that the functional selectivity of such agents may arise from a bitopic mechanism.

ARAP3 is transiently tyrosine phosphorylated in cells attaching to fibronectin and inhibits cell spreading in a RhoGAP-dependent manner

ARAP3 is a GTPase activating protein (GAP) for Rho and Arf GTPases that is implicated in phosphoinositide 3-kinase (PI 3-kinase) signalling pathways controlling lamellipodia formation and actin stress fibre assembly. We have identified ARAP3 as a phosphorylated target of protein tyrosine kinases. In cells, ARAP3 was tyrosine phosphorylated when co-expressed with Src-family kinases (SFKs), upon stimulation with growth factors and during adhesion to the extracellular matrix (ECM) substrate fibronectin. Adhesion-induced phosphorylation of ARAP3 was suppressed by selective inhibitors of Src-family kinases and PI 3-kinase and by a Src dominant interfering mutant. Inducible expression of ARAP3 in HEK293 epithelial cells resulted in increased cell rounding, membrane process formation and cell clustering on ECM substrates. In contrast, ARAP3 dramatically slowed the kinetics of cell spreading on fibronectin but had no effect on cell adhesion. These effects of ARAP3 required a functional Rho GAP domain and were associated with reduced cellular levels of active RhoA and Rac1 but did not require the sterile alpha motif (SAM) or Arf GAP domains. Mutation of two phosphorylation sites, Y1399 and Y1404, enhanced some ARAP3 activities, suggesting that ARAP3 may be negatively regulated by phosphorylation on these tyrosine residues. These results implicate ARAP3 in integrin-mediated tyrosine kinase signalling pathways controlling Rho GTPases and cell spreading.

Autophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (ttis450), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In ttis450, the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in ttis450 larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in ttis450 larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.

Distinct requirements for the Sprouty domain for functional activity of Spred proteins

Sprouty and Spred {Sprouty-related EVH1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] domain} proteins have been identified as antagonists of growth factor signalling pathways. We show here that Spred-1 and Spred-2 appear to have distinct mechanisms whereby they induce their effects, as the Sprouty domain of Spred-1 is not required to block MAPK (mitogen-activated protein kinase) activation, while that of Spred-2 is required. Similarly, deletion of the C-terminal Sprouty domain of Spred-1 does not affect cell-cycle progression of G(0)-synchronized cells through to S-phase following growth factor stimulation, while the Sprouty domain is required for Spred-2 function. We also demonstrate that the inhibitory function of Spred proteins is restricted to the Ras/MAPK pathway, that tyrosine phosphorylation is not required for this function, and that the Sprouty domain mediates heterodimer formation of Spred proteins. Growth-factor-mediated activation of the small GTPases, Ras and Rap1, was able to be regulated by Spred-1 and Spred-2, without affecting receptor activation. Taken together, these results highlight the potential for different functional roles of the Sprouty domain within the Spred family of proteins, suggesting that Spred proteins may use different mechanisms to induce inhibition of the MAPK pathway.

p50 Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

ABSTRACT Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast. We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50Cdc37, the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50Cdc37 in the folding and stabilization of Src family kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50Cdc37 rescues the catalytic activity of tsHck499F at 33°C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39°C). Hsp90 function is required for tsHck499F activity and its stabilization by p50Cdc37, but overexpression of Hsp90 is not sufficient to stabilize tsHck499F. Overexpression of p50Cdc37 promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50Cdc37might be the rate-limiting step in the association oftsHck499F with Hsp90. A truncation mutant of p50Cdc37 that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50Cdc37 may also act allosterically to enhance the association of tsHck499F with Hsp90. The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.

Determination of the Disulfide Structure andN-Glycosylation Sites of the Extracellular Domain of the Human Signal Transducer gp130

gp130 is the common signal transducing receptor subunit for the interleukin-6-type family of cytokines. Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys279 and Cys469), and one (Cys397) is modified byS-cysteinylation. Of the 11 potentialN-glycosylation sites, Asn21, Asn61, Asn109, Asn135, Asn205, Asn357, Asn361, Asn531, and Asn542 are glycosylated but not Asn224 and Asn368. The disulfide bonds, Cys112–Cys122 and Cys150–Cys160, are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys6–Cys32 and Cys26–Cys81) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys436–Cys444. We have created a model predicting that this loop maintains Cys469 in a reduced form, available for ligand-induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130.

G2(MP2,SVP) study of the relationship between the benzyl and tropyl radicals, and their cation analogues

Abstract The calculated heat of formation and ionization energy of the benzyl radical at the G2(MP2,SVP) level differ from recent experimental estimates by just 1 kJ mol −1 . The ionization energy of the tropyl radical calculated at this level differs from experiment by 5 kJ mol −1 . The heat of formation of tropyl, 281 ± 8 kJ mol −1 , is significantly higher than experimental estimates, suggesting the need for experimental re-evaluation. The lowest energy path for rearrangement of the benzyl cation to tropylium proceeds through a single intermediate minimum, the 1-cycloheptatrienyl cation, and requires 272 kJ mol −1 .

Disulfide Bond Structure and N-Glycosylation Sites of the Extracellular Domain of the Human Interleukin-6 Receptor

The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular “soluble” part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potentialN-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226.

Exploring the utility of human DNA methylation arrays for profiling mouse genomic DNA

Illumina Infinium Human Methylation (HM) BeadChips are widely used for measuring genome-scale DNA methylation, particularly in relation to epigenome-wide association studies (EWAS) studies. The methylation profile of human samples can be assessed accurately and reproducibly using the HM27 BeadChip (27,578 CpG sites) or its successor, the HM450 BeadChip (482,421 CpG sites). To date no mouse equivalent has been developed, greatly hindering the application of this methodology to the wide range of valuable murine models of disease and development currently in existence. We found 1308 and 13,715 probes from HM27 and HM450 BeadChip respectively, uniquely matched the bisulfite converted reference mouse genome (mm9). We demonstrate reproducible measurements of DNA methylation at these probes in a range of mouse tissue samples and in a murine cell line model of acute myeloid leukaemia. In the absence of a mouse counterpart, the Infinium Human Methylation BeadChip arrays have utility for methylation profiling in non-human species.