Agonist-induced trafficking of the low-affinity formyl peptide receptor FPRL1

The formyl peptide-like receptor FPRL1 is a member of the chemoattractant subfamily of G protein- coupled receptors involved in regulating leukocyte migration in inflammation. To elucidate mechanisms underlying the internalization of ligand-bound FPRL1 and possible receptor recycling, we characterized the endocytic itinerary of FPRL1. We show that agonist-triggered internalization from the plasma membrane into intracellular compartments is prevented by perturbation of clathrin-mediated endocytosis, such as expression of the dominant-negative clathrin Hub mutant, siRNA-mediated depletion of cellular clathrin and expression of a dominant-negative mutant of the large GTPase dynamin. Internalized FPRL1 co-localized with endocytosed transferrin and the small GTPases Rab4 and Rab11 in vesicular structures most resembling recycling endosomes. Recycling of FPRL1 was significantly reduced by pretreatment with PI3-kinase inhibitors. Thus, ligand-bound FPRL1 undergoes primarily clathrin-mediated and dynamin-dependent endocytosis and the receptor recycles via a rapid PI3-kinase-sensitive route as well as pathways involving perinuclear recycling endosomes.Received 19 March 2004; received after revision 26 April 2004; accepted 12 May 2004     

Molecular recognition of a single sphingolipid species by a protein's transmembrane domain

Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.     

Crystal structure of metarhodopsin II

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta?II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta?II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta?II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0?? and 2.85?? crystal structures, respectively, of Meta?II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta?II structures, the electron density from the retinal ligand seamlessly continues into the Lys?296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta?II. The structures can now serve as models for the large GPCR family.     

A luminous quasar at a redshift of z = 7.085

The intergalactic medium was not completely reionized until approximately a billion years after the Big Bang, as revealed by observations of quasars with redshifts of less than 6.5. It has been difficult to probe to higher redshifts, however, because quasars have historically been identified in optical surveys, which are insensitive to sources at redshifts exceeding 6.5. Here we report observations of a quasar (ULAS?J112001.48+064124.3) at a redshift of 7.085, which is 0.77 billion years after the Big Bang. ULAS?J1120+0641 has a luminosity of 6.3 × 10(13)L(⊙) and hosts a black hole with a mass of 2 × 10(9)M(⊙) (where L(⊙) and M(⊙) are the luminosity and mass of the Sun). The measured radius of the ionized near zone around ULAS?J1120+0641 is 1.9?megaparsecs, a factor of three smaller than is typical for quasars at redshifts between 6.0 and 6.4. The near-zone transmission profile is consistent with a Lyα damping wing, suggesting that the neutral fraction of the intergalactic medium in front of ULAS?J1120+0641 exceeded 0.1.     

A high-resolution map of human evolutionary constraint using 29 mammals

The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ～4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ～60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease.     

Emerging local Kondo screening and spatial coherence in the heavy-fermion metal YbRh2Si2

The entanglement of quantum states is both a central concept in fundamental physics and a potential tool for realizing advanced materials and applications. The quantum superpositions underlying entanglement are at the heart of the intricate interplay of localized spin states and itinerant electronic states that gives rise to the Kondo effect in certain dilute magnetic alloys. In systems where the density of localized spin states is sufficiently high, they can no longer be treated as non-interacting; if they form a dense periodic array, a Kondo lattice may be established. Such a Kondo lattice gives rise to the emergence of charge carriers with enhanced effective masses, but the precise nature of the coherent Kondo state responsible for the generation of these heavy fermions remains highly debated. Here we use atomic-resolution tunnelling spectroscopy to investigate the low-energy excitations of a generic Kondo lattice system, YbRh(2)Si(2). We find that the hybridization of the conduction electrons with the localized 4f electrons results in a decrease in the tunnelling conductance at the Fermi energy. In addition, we observe unambiguously the crystal-field excitations of the Yb(3+) ions. A strongly temperature-dependent peak in the tunnelling conductance is attributed to the Fano resonance resulting from tunnelling into the coherent heavy-fermion states that emerge at low temperature. Taken together, these features reveal how quantum coherence develops in heavy 4f-electron Kondo lattices. Our results demonstrate the efficiency of real-space electronic structure imaging for the investigation of strong electronic correlations, specifically with respect to coherence phenomena, phase coexistence and quantum criticality.     

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