Heini M. Miettinen

IDENTIFICATION OF A LIGAND BINDING SITE IN THE HUMAN NEUTROPHIL FORMYL PEPTIDE RECEPTOR USING A SITE-SPECIFIC FLUORESCENT PHOTOAFFINITY LABEL AND MASS SPECTROMETRY

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-l-phenylalanine-Phe-Tyr-Lys-ε-N-fluorescein (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83–87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83–85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83–85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol.159, 4045–4054) and the photochemical cross-linking data.

Self-assembling biomolecular catalysts for hydrogen production

The chemistry of highly evolved protein-based compartments has inspired the design of new catalytically active materials that self-assemble from biological components. A frontier of this biodesign is the potential to contribute new catalytic systems for the production of sustainable fuels, such as hydrogen. Here, we show the encapsulation and protection of an active hydrogen-producing and oxygen-tolerant [NiFe]-hydrogenase, sequestered within the capsid of the bacteriophage P22 through directed self-assembly. We co-opted Escherichia coli for biomolecular synthesis and assembly of this nanomaterial by expressing and maturing the EcHyd-1 hydrogenase prior to expression of the P22 coat protein, which subsequently self assembles. By probing the infrared spectroscopic signatures and catalytic activity of the engineered material, we demonstrate that the capsid provides stability and protection to the hydrogenase cargo. These results illustrate how combining biological function with directed supramolecular self-assembly can be used to create new materials for sustainable catalysis.

Characterization of the Binding Site on the Formyl Peptide Receptor Using Three Receptor Mutants and Analogs of Met-Leu-Phe and Met-Met-Trp-Leu-Leu

The formyl peptide receptor (FPR) is a chemotactic G protein-coupled receptor found on the surface of phagocytes. We have previously shown that the formyl peptide binding site maps to the membrane-spanning region (Miettinen, H. M., Mills, J. S., Gripentrog, J. M., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045–4054). Recent reports have indicated that non-formylated peptides, such as MMWLL can also activate this receptor (Chen, J., Bernstein, H. S., Chen, M., Wang, L., Ishi, M., Turck, C. W., and Coughlin, S. R. (1995) J. Biol. Chem. 270, 23398–23401.) Here we show that the selectivity for the binding of different NH2-terminal analogs of MMWLL or MLF can be markedly altered by mutating Asp-106 to asparagine or Arg-201 to alanine. Both D106N and R201A produced a similar change in ligand specificity, including an enhanced ability to bind the HIV-1 peptide DP178. In contrast, the mutation R205A exhibited altered specificity at the COOH terminus of fMLF, with R205A binding fMLF-O-butyl >fMLF-O-methyl > fMLF, whereas wt FPR bound fMLF >fMLF-O-methyl ∼fMLF-O-butyl. These data, taken together with our previous finding that the leucine side chain of fMLF is probably bound to FPR near FPR 93VRK95 (Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., and Jesaitis, A. J. (1998)J. Biol. Chem. 273, 10428–10435.), indicate that the most likely positioning of fMLF in the binding pocket of FPR is approximately parallel to the fifth transmembrane helix with the formamide group of fMLF hydrogen-bonded to both Asp-106 and Arg-201, the leucine side chain pointing toward the second transmembrane region, and the COOH-terminal carboxyl group offMLF ion-paired with Arg-205.

Identification of putative sites of interaction between the human formyl peptide receptor and G protein.

Wild-type and 35 mutant formyl peptide receptors (FPRs) were stably expressed in Chinese hamster ovary cells. All cell surface-expressed mutant receptors bound N-formyl peptide with similar affinities as wild-type FPR, suggesting that the mutations did not affect the ligand-binding site. G protein coupling was examined by quantitative analysis ofN-formyl-methionyl-leucyl-phenylalanine-induced increase in binding of 35S-labeled guanosine 5′-3-O-(thio)triphosphate (GTPγS) to membranes. The most prominent uncoupled FPR mutants were located in the N-terminal part of the second transmembrane domain (S63W and D71A) and the C-terminal interface of the third transmembrane domain (R123A and C124S/C126S). In addition, less pronounced uncoupling was detected with deletion mutations in the third cytoplasmic loop and in the cytoplasmic tail. Further analysis of some of the mutants that were judged to be uncoupled based on the [35S]GTPγS membrane-binding assay were found to transduce a signal, as evidenced by intracellular calcium mobilization and activation of p42/44 MAPK. Thus, these single point mutations in FPR did not completely abolish the interaction with G protein, emphasizing that the coupling site is coordinated by several different regions of the receptor. Mutations located in the putative fifth and sixth transmembrane domains near the N- and C-terminal parts of the third cytoplasmic loop did not result in uncoupling. These regions have previously been shown to be critical for G protein coupling to many other G protein-coupled receptors. Thus, FPR appears to have a G protein-interacting site distinct from the adrenergic receptors, the muscarinic receptors, and the angiotensin receptors.

Different Endocytosis Pathways of the C5a Receptor and the N-formyl Peptide Receptor

Two chemoattractant receptors, C5aR (the complement fragment C5a receptor) and FPR (the N‐formyl peptide receptor), are involved in neutrophil activation at sites of inflammation. In this study, we found major differences in the intracellular trafficking of the receptors in transfected Chinese hamster ovary (CHO) cells. Western blot analysis showed that FPR was stable during a 3 h stimulation with ligand, but C5aR was reduced in quantity by 50%. Not all C5aR was targeted directly for degradation however; a small, but visible fraction of the receptor became re‐phosphorylated upon subsequent addition of ligand, suggesting that some of the receptor had cycled to the cell surface. Light membrane fractions isolated from activated cells showed C5aR distribution at the bottom of a glycerol gradient, colocalizing with the main distribution of the late endosomal/lysosomal marker LAMP2, whereas FPR was found at the bottom of the gradient as well as in the middle of the gradient, where it cofractionated with the early/sorting endosomal marker Rab5. Using fluorescence microscopy, we observed ligand‐dependent redistribution of C5aR‐EGFP from the plasma membrane to LAMP2‐positive compartments, whereas FPR‐EGFP showed significant colocalization with the early/sorting endosomes. Analysis of endogenous C5aR and FPR in neutrophils revealed a pattern similar to the CHO transfectants: C5aR underwent degradation after prolonged ligand stimulation, while FPR did not. Finally, we confirmed the down‐regulation of C5aR in a functional assay by showing reduced chemotaxis toward C5a in both CHO transfectants and neutrophils after preincubation with C5a. A similar decrease in FPR‐mediated chemotaxis was not observed.

Receptor tyrosine kinase and G-protein coupled receptor signaling and sorting within endosomes

*DivisionofBiologicalSciencesandCenterforStructuralandFunctionalNeuroscience,UniversityofMontana, Missoula, Montana, USA Department of Microbiology, Montana State University, Bozeman, Montana, USAMany neuroscientists desire to understand events that occurat the synapse. We would like to know in exacting detail howreceptors respond to neurotransmitters and neuropeptides.Unfortunately, some mechanisms such as receptors’ mem-brane traffic and signal transduction are not accessible orpractically studied at synapses in vivo, so model systemsmust be used.Data gleaned invitrofrom neuronsor cell linesin culture using the tools ofcell biology has great value infocusing our views about how receptors behave in vivo;nature is conservative and once mechanisms and rules ofbehavior are established they usually hold true in manydifferent contexts. This review examines the hypothesis thatendosomes form organelles that are specialized for signaltransduction, and casts a wide net fishing for clues from cellbiologyabouthowreceptorsmight dothisataxontips.Manyofthese clues are derived from molecular interactions amonggene products with known roles in signal transduction,membrane traffic, and the cytoskeleton.Not so long ago, investigators who studied signaltransduction, membrane traffic, or the cytoskeleton focusedtheir attention mostly within these individual disciplines.Recently these formerly distinct fields have become linkedtogether in surprising and marvelous ways. Endocytosis isregulated by signal transduction and signal transduction isinitiated from endosomes. The first evidence that receptorinternalization is not solely a mechanism for down-regula-tion and termination ofthe receptor’s signal, that receptorscan signal from endosomes, was provided for epidermalgrowth factor (EGF) and insulin receptors by Bergeron andcolleagues (Baass et al. 1995). It is now widely recognizedthat signaling from both receptor tyrosine kinases (RTKs)and G-protein coupled receptors (GPCRs) occurs in intra-cellular organelles derived from endocytosis (Ceresa andSchmid 2000; Di Fiore and De Camilli 2001; Ferguson2001; McPherson et al. 2001; Wiley and Burke 2001).Receptor signal transduction from intracellular organellesactivates distinct pathways and conveys instructions tospecific intracellular locations. In neurons, signaling afterendocytosis is a compelling means to convey neurotrophinsignals from the tips of axons to the cell body.RTK signaling from endocytic organellesand the signaling vesicles hypothesisDuring development ofthe nervous system, more neuronsare born than are needed and in mammals there is a selectionfor those which make functional connections. Neurons thatdo not make proper connections activate programmed celldeath; neurons that do connect survive and differentiate.When projecting axons reach their target they receiveinstructions in the form of polypeptide growth factors,including neurotrophins such as nerve growth factor (NGF),which bind to receptors (Trks) at the axon tip. This distalsignal must traverse down the axon, a great distance in somecases, to direct the cell body towards differentiation andaway from programmed cell death. Defects in initiation,reception, or transport ofneurotrophin signals may lead tomany different diseases of congenital cognitive dysfunctionand neurodegeneration (Cooper et al. 2001; Sofroniew .2001). It is thus ofvital importance to understand inmolecular detail the generation, conveyance, intracellularlocation, and downstream targets ofneurotrophin signals.One indication ofthe perceived importance ofneurotrophinsin the development andmaintenance ofthe nervous system isthe number ofrecent reviews covering this field, no fewer

Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9.

Experimental Evidence for Lack of Homodimerization of the G Protein-Coupled Human N-Formyl Peptide Receptor

A large number of G protein-coupled receptors have been shown to form homodimers based on a number of different techniques such as receptor coimmunoprecipitation, cross-linking, and fluorescence resonance energy transfer. In addition, functional assays of cells coexpressing a mutant receptor with a wild-type receptor have shown receptor phenotypes that can best be explained through dimerization. We asked whether the human neutrophil N-formyl peptide receptor (FPR) forms dimers in Chinese hamster ovary cells by coexpressing wild-type FPR with one of two mutants: D71A, which is uncoupled from G protein, and N297A, which has a defect in receptor phosphorylation and endocytosis. Experiments measuring chemotaxis, ligand-induced release of intracellular calcium, and p42/44 mitogen-activated protein kinase activation did not show an inhibitory effect of the coexpressed FPR D71A mutant. Coexpressed wild-type receptor was efficiently internalized, but failed to correct the endocytosis defects of the D71A and the N297A mutants. To explore the possibility that the mutations themselves prevented dimerization, we examined the coimmunoprecipitation of differentially epitope-tagged FPR. Immunoprecipitation of hemagglutinin-tagged FPR failed to coimmunoprecipitate coexpressed c-myc-tagged FPR and vice versa. Together, these data suggest that, unlike many other G protein-coupled receptors, FPR does not form homodimers.

C-Terminal Tail Phosphorylation of N-Formyl Peptide Receptor: Differential Recognition of Two Neutrophil Chemoattractant Receptors by Monoclonal Antibodies NFPR1 and NFPR2

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni2+-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45–75 kDa species (peak Mr ∼60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of Mr ∼40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting ∼60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337–346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.

The N-Formyl Peptide Receptor

The chemotaxis of phagocytic leukocytes from the blood to tissues identifies the acute (neutrophil) and chronic (macrophages) inflammatory condition (1). This hallmark process is central to host defense against microorganisms, to mediation of the immune response, and to repair of injured tissues. When unregulated, however, it is also the key vehicle of tissue damage and injury. Consequently, leukocyte chemotaxis, its ligands, and its receptors continue to be the focus of intense study. With a comprehensive molecular understanding of the structure-function relationships of chemotactic activation, intervention that mitigates injury but enhances microbial killing may become possible.