In the pancreas, Notch signaling is thought to prevent cell differentiation, thereby maintaining progenitors in an undifferentiated state. Here, we show that Notch renders progenitors competent to differentiate into ductal and endocrine cells by inducing activators of cell differentiation. Notch signaling promotes the expression of Sox9, which cell-autonomously activates the pro-endocrine gene Ngn3. However, at high Notch activity endocrine differentiation is blocked, as Notch also induces expression of the Ngn3 repressor Hes1. At the transition from high to intermediate Notch activity, only Sox9, but not Hes1, is maintained, thus de-repressing Ngn3 and initiating endocrine differentiation. In the absence of Sox9 activity, endocrine and ductal cells fail to differentiate, resulting in polycystic ducts devoid of primary cilia. Although Sox9 is required for Ngn3 induction, endocrine differentiation necessitates subsequent Sox9 downregulation and evasion from Notch activity via cell-autonomous repression of Sox9 by Ngn3. If high Notch levels are maintained, endocrine progenitors retain Sox9 and undergo ductal fate conversion. Taken together, our findings establish a novel role for Notch in initiating both ductal and endocrine development and reveal that Notch does not function in an on-off mode, but that a gradient of Notch activity produces distinct cellular states during pancreas development.
G protein-coupled receptors (GPCRs) activate four families of heterotrimeric G proteins, and individual receptors must select a subset of G proteins to produce appropriate cellular responses. Although the precise mechanisms of coupling selectivity are uncertain, the Gα subunit C terminus is widely believed to be the primary determinant recognized by cognate receptors. Here, we directly assess coupling between 14 representative GPCRs and 16 Gα subunits, including one wild-type Gα subunit from each of the four families and 12 chimeras with exchanged C termini. We use a sensitive bioluminescence resonance energy transfer (BRET) assay that provides control over both ligand and nucleotide binding, and allows direct comparison across G protein families. We find that the Gs- and Gq-coupled receptors we studied are relatively promiscuous and always couple to some extent to Gi1 heterotrimers. In contrast, Gi-coupled receptors are more selective. Our results with Gα subunit chimeras show that the Gα C terminus is important for coupling selectivity, but no more so than the Gα subunit core. The relative importance of the Gα subunit core and C terminus is highly variable and, for some receptors, the Gα core is more important for selective coupling than the C terminus. Our results suggest general rules for GPCR-G protein coupling and demonstrate that the critical G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein.
To investigate the fidelity of canonical non-homologous end joining (C-NHEJ), we developed an assay to detect EJ between distal ends of two Cas9-induced chromosomal breaks that are joined without causing insertion/deletion mutations (indels). Here we find that such EJ requires several core C-NHEJ factors, including XLF. Using variants of this assay, we find that C-NHEJ is required for EJ events that use 1–2, but not ≥3, nucleotides of terminal microhomology. We also investigated XLF residues required for EJ without indels, finding that one of two binding domains is essential (L115 or C-terminal lysines that bind XRCC4 and KU/DNA, respectively), and that disruption of one of these domains sensitizes XLF to mutations that affect its dimer interface, which we examined with molecular dynamic simulations. Thus, C-NHEJ, including synergistic function of distinct XLF domains, is required for EJ of chromosomal breaks without indels.
While the dynamics of the intracellular surface in agonist-stimulated GPCRs is well studied, the impact of GPCR dynamics on G-protein selectivity remains unclear. Here, we combine molecular dynamics simulations with live-cell FRET and secondary messenger measurements, for 21 GPCR−G-protein combinations, to advance a dynamic model of the GPCR−G-protein interface. Our data show C terminus peptides of Gα s , Gα i , and Gα q proteins assume a small ensemble of unique orientations when coupled to their cognate GPCRs, similar to the variations observed in 3D structures of GPCR−G-protein complexes. The noncognate G proteins interface with latent intracellular GPCR cavities but dissociate due to weak and unstable interactions. Three predicted mutations in β 2 -adrenergic receptor stabilize binding of noncognate Gα q protein in its latent cavity, allowing promiscuous signaling through both Gα s and Gα q in a dose-dependent manner. This demonstrates that latent GPCR cavities can be evolved, by design or nature, to tune G-protein selectivity, giving insights to pluridimensional GPCR signaling.G-protein−coupled receptor | GPCR | functional selectivity | structural plasticity | dynamics Author contributions: M.S.
Although the importance of the C terminus of the ␣ subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the G␣ subunit (G␣ peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between G␣ peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate G␣ peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate G␣ peptide stabilizes the agonistbound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (G␣ s /G␣ q -Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the 2-adrenergic receptor (2-AR)-G␣ s and V 1A receptor (V 1A R)-G␣ q . The G␣ s and G␣ q peptides adopt different orientations in 2-AR and V 1A R, respectively. The 2-AR/G␣ s peptide interface is dominated by electrostatic interactions, whereas the V 1A R/G␣ q peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in G␣ q prevents this peptide from interacting strongly with 2-AR. Mutagenesis to the G␣ s counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.In recent years, there has been significant progress in structural and spectroscopic studies of Class A G protein-coupled receptors (GPCR), 3 which are energizing structure-based drug discovery efforts (1-8). Although these studies clearly demonstrate ligand-dependent structural changes in the GPCR, there remains a paucity of information on how GPCR conformation translates to selective G protein activation (9). Without structural information to compare and contrast multiple GPCR/G protein interfaces, the underlying mechanisms of selection remain incompletely understood. Currently, there is only one crystal structure of the complete GPCR/G protein interface (2-AR⅐G s complex) that provides a single essential snapshot of a highly dynamic interaction (10). Hence, alternative approaches are essential to dissect the structural elements within both the GPCR and its effectors that confer signaling specificity within the cellular environment. In this study, we combine FRETbased measurements of the GPCR/G protein interface in live cells, combined with computational modeling to build a rational scalable approach to identify structural hot spots that drive effector selection.One critical and well characterized component of the GPCR/G prote...
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