Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations

G protein–coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous β2-adrenergic receptor (β2AR) and muscarinic acetylcholine M3 receptor (M3R) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal–regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations

Ligand-dependent spatiotemporal signaling profiles of the mu-opioid receptor are controlled by distinct protein-interaction networks

Ligand-dependent differences in the regulation and internalization of the mu-opioid receptor (MOR) have been linked to the severity of adverse effects that limit opiate use in pain management. MOR activation by morphine or [D-Ala2,N-MePhe4,Gly-ol]-enkephalin (DAMGO) causes differences in spatiotemporal signaling dependent on MOR distribution at the plasma membrane. Morphine stimulation of MOR activates a Gai/o–Gbg–protein kinase C (PKC)a phosphorylation pathway that limits MOR distribution and is associated with a sustained increase in cytosolic extracellular signal–regulated kinase (ERK) activity. In contrast, DAMGO causes a redistribution of the MOR at the plasma membrane (before receptor internalization), that facilitates transient activation of cytosolic and nuclear ERK. Here, we used proximity biotinylation proteomics to dissect the different protein-interaction networks that underlie the spatiotemporal signaling of morphine and DAMGO. We found that DAMGO, but not morphine, activates Ras‐related C3 botulinum toxin substrate 1 (Rac1). Both Rac1 and nuclear ERK activity was dependent on the scaffolding proteins IQ motif–containing GTPase-activating protein-1 (IQGAP1) and Crk-like protein (CRKL). In contrast, morphine increased the proximity of the MOR to desmosomal proteins, which form specialized and highly ordered membrane domains. Knockdown of two desmosomal proteins, junction plakoglobin (JUP) or desmocolin-1 (DSC1), switched the morphine spatiotemporal signaling profile to mimic that of DAMGO, resulting in a transient increase in nuclear ERK activity. The identification of the MOR-interaction networks that control differential spatiotemporal signaling reported here is an important step towards understanding how signal compartmentalization contributes to opioid-induced responses including anti-nociception and the development of tolerance and dependence

The phosphatidylinositol (3,4,5)-trisphosphate-dependent rac exchanger 1:Ras-related C3 botulinum toxin substrate 1 (P-Rex1:Rac1) complex reveals the basis of Rac1 activation in breast cancer cells

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP(3))-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP(3) and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP(3)/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP(3) and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site

The Structural Impact of a Polyglutamine Tract Is Location-Dependent

Polyglutamine (polyQ) expansion leads to protein aggregation and neurodegeneration in Huntington's disease and eight other inherited neurological conditions. Expansion of the polyQ tract beyond a threshold of 37 glutamines leads to the formation of toxic nuclear aggregates. This suggests that polyQ expansion causes a conformational change within the protein, the nature of which is unclear. There is a trend in the disease proteins that the polyQ tract is located external to but not within a structured domain. We have created a model polyQ protein in which the repeat location mimics the flexible environment of the polyQ tract in the disease proteins. Our model protein recapitulates the aggregation features observed with the clinical proteins and allows structural characterization. With the use of NMR spectroscopy and a range of biophysical techniques, we demonstrate that polyQ expansion into the pathological range has no effect on the structure, dynamics, and stability of a domain adjacent to the polyQ tract. To explore the clinical significance of repeat location, we engineered a variant of the model protein with a polyQ tract within the domain, a location that does not mimic physiological context, demonstrating significant destabilization and structural perturbation. These different effects highlight the importance of repeat location. We conclude that protein misfolding within the polyQ tract itself is the driving force behind the key characteristics of polyQ disease, and that structural perturbation of flanking domains is not required

Structure of the complete human TSC:WIPI3 lysosomal recruitment complex

Tuberous sclerosis complex (TSC) turns off cell growth in response to energy stress by inhibiting the master kinase mechanistic target of rapamycin complex (mTORC1). TSC hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, preventing the allosteric activation of mTORC1. Loss-offunction TSC mutations hyperactivate mTORC1 resulting in the common genetic disorder TSC characterized by excess cell growth and tumor formation. Here we overcome a high degree of continuous conformational heterogeneity to determine the 2.9 Å cryo-electron microscopy (cryo-EM) structure of the complete human TSC in complex with the lysosomal recruitment factor WIPI3. TSC forms an elongated 40 nm wing-like structure with a core HEAT-repeat scaffold formed by a TSC2 dimer joined centrally by the juxtaposition of two catalytic domains. The TSC1 coil-coil dimer runs across the TSC2 surface, forming a previously undetected N-terminal TSC1 dimer that clamps onto the core scaffold on a single TSC wing. Structural and biochemical analysis reveals a novel phosphatidylinositol phosphate (PIP)-binding pocket in the TSC1 dimer interface that specifically binds singularly phosphorylated PIPs. WD repeat domain phosphoinositide-interacting-protein-3 (WIPI3) binds to the extreme tip of the complex through a conserved motif in TSC1, providing a second membrane anchor point for TSC lysosomal recruitment. The TSC:WIPI3 complex helps explain how TSC lysosomal recruitment proteins coordinate with endolysosomal phosphoinositide-signaling networks to regulate TSC localization, RHEB hydrolysis, and mTORC1 inhibition. More broadly, the high-resolution structure of the complete human TSC identifies novel mutational hotspots that unravel crucial new mechanisms of TSC dysregulation in disease.</p

Stonefish toxin defines an ancient branch of the perforin-like superfamily

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom

Cell Traversal Activity Is Important for Plasmodium falciparum Liver Infection in Humanized Mice

Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparum sporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparum growth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparum sporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen