Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4)

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.     

Noncanonical Role of the PDZ4 Domain of the Adaptor Protein PDZK1 in the Regulation of the Hepatic High Density Lipoprotein Receptor Scavenger Receptor Class B,Type I (SR-BI)

The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI''s cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1''s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.     

Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1

The plant hormone ethylene plays important roles in growth and development. Ethylene is perceived by a family of membrane-bound receptors that actively repress ethylene responses. When the receptors bind ethylene, their signaling is shut off, activating responses. REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) encodes a novel membrane protein conserved in plants and metazoans. Genetic analyses in Arabidopsis thaliana suggest that RTE1 promotes the signaling state of the ethylene receptor ETR1 through the ETR1 N-terminal domain. RTE1 and ETR1 have been shown to co-localize to the endoplasmic reticulum (ER) and Golgi apparatus in Arabidopsis. Here, we demonstrate a physical association of RTE1 and ETR1 using in vivo and in vitro methods. Interaction of RTE1 and ETR1 was revealed in vivo by bimolecular fluorescence complementation (BiFC) in a tobacco cell transient assay and in stably transformed Arabidopsis. The association was also observed using a truncated version of ETR1 comprising the N terminus (amino acids 1-349). Interaction of RTE1 and ETR1 was confirmed by co-immunoprecipitation from Arabidopsis. The interaction occurs with high affinity (K(d), 117 nM) based on tryptophan fluorescence spectroscopy using purified recombinant RTE1 and a tryptophan-less version of purified recombinant ETR1. An amino acid substitution (C161Y) in RTE1 that is known to confer an ETR1 loss-of-function phenotype correspondingly gives a nearly 12-fold increase in the dissociation constant (K(d), 1.38 μM). These findings indicate that a high affinity association of RTE1 and ETR1 is important in the regulation of ETR1.     

Structure, Affinity, and Availability of Estrogen Receptor Complexes in the Cellular Environment

An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated Förster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor α-isoform (ERα) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ERα with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ERαs at low free SRC-RID concentrations (<2 nm) to lower affinity complexes with an ERα monomer at higher SRC-RID concentrations (∼10 nm). The methods also showed that only a subpopulation of ERα was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment.     

Post-synaptic Density-95 (PSD-95) Binding Capacity of G-protein-coupled Receptor 30 (GPR30), an Estrogen Receptor That Can Be Identified in Hippocampal Dendritic Spines

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.     

Structural Determinants for Binding of Sorting Nexin 17 (SNX17) to the Cytoplasmic Adaptor Protein Krev Interaction Trapped 1 (KRIT1)

Sorting nexin 17 (SNX17) is a member of the family of cytoplasmic sorting nexin adaptor proteins that regulate endosomal trafficking of cell surface proteins. SNX17 localizes to early endosomes where it directly binds NPX(Y/F) motifs in the cytoplasmic tails of its target receptors to mediate their rates of endocytic internalization, recycling, and/or degradation. SNX17 has also been implicated in mediating cell signaling and can interact with cytoplasmic proteins. KRIT1 (Krev interaction trapped 1), a cytoplasmic adaptor protein associated with cerebral cavernous malformations, has previously been shown to interact with SNX17. Here, we demonstrate that SNX17 indeed binds directly to KRIT1 and map the binding to the second Asn-Pro-Xaa-Tyr/Phe (NPX(Y/F)) motif in KRIT1. We further characterize the interaction as being mediated by the FERM domain of SNX17. We present the co-crystal structure of SNX17-FERM with the KRIT1-NPXF2 peptide to 3.0 Å resolution and demonstrate that the interaction is highly similar in structure and binding affinity to that between SNX17 and P-selectin. We verify the molecular details of the interaction by site-directed mutagenesis and pulldown assay and thereby confirm that the major binding site for SNX17 is confined to the NPXF2 motif in KRIT1. Taken together, our results verify a direct interaction between SNX17 and KRIT1 and classify KRIT1 as a SNX17 binding partner.     

Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of membrane attack complex (MAC)

The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8αβγ heterodimer and perforin show that the central domain of C6 adopts a "closed" (perforin-like) state that is distinct from the "open" conformations in C8. We further show that C6, C8α, and C8β contain three homologous subdomains ("upper," "lower," and "regulatory") related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8β, rotation of the regulatory segment is linked to an opening of the central β-sheet of its clockwise partner, C8α. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the β-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin.     

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

Flp-In^TM T-REx^TM 293 cells expressing a wild type human M₃ muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M₃ receptor expressed stably in Flp-In^TM TREx^TM 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC₅₀ values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M₃ muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.     

A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.     

Identification of intersubunit domain interactions within eukaryotic initiation factor (eIF) 2B, the nucleotide exchange factor for translation initiation

In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α-ε) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ε that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bε for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bε and -γ PLDs and a shared second domain predicted to form a left-handed β helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed β helix domains that form the eIF2Bγε subcomplex and propose a model for domain interactions between eIF2B subunits.     

Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor: A REGION CRITICAL FOR BIOLOGICAL ACTIVITY*

Understanding the molecular basis of natural ligand binding and activation of the glucagon-like peptide 1 (GLP1) receptor may facilitate the development of agonist drugs useful for the management of type 2 diabetes mellitus. We previously reported molecular approximations between carboxyl-terminal residues 24 and 35 within GLP1 and its receptor. In this work, we have focused on the amino-terminal region of GLP1, known to be critical for receptor activation. We developed two high-affinity, full agonist photolabile GLP1 probes having sites of covalent attachment in positions 6 and 12 of the 30-residue peptide (GLP1(7–36)). Both probes bound to the receptor specifically and covalently labeled single distinct sites. Chemical and protease cleavage of the labeled receptor identified the juxtamembrane region of its amino-terminal domain as the region of covalent attachment of the position 12 probe, whereas the region of labeling by the position 6 probe was localized to the first extracellular loop. Radiochemical sequencing identified receptor residue Tyr¹⁴⁵, adjacent to the first transmembrane segment, as the site of labeling by the position 12 probe, and receptor residue Tyr²⁰⁵, within the first extracellular loop, as the site of labeling by the position 6 probe. These data provide support for a common mechanism for natural ligand binding and activation of family B G protein-coupled receptors. This region of interaction of peptide amino-terminal domains with the receptor may provide a pocket that can be targeted by small molecule agonists.     

Identification and Analysis of a Novel Dimerization Domain Shared by Various Members of c-Jun N-terminal Kinase (JNK) Scaffold Proteins

Mitogen-activated protein kinases (MAPKs) form a kinase tier module in which MAPK, MAP2K, and MAP3K are held by scaffold proteins. The scaffold proteins serve as a protein platform for selective and spatial kinase activation. The precise mechanism by which the scaffold proteins function has not yet been fully explained. WDR62 is a novel scaffold protein of the c-Jun N-terminal kinase (JNK) pathway. Recessive mutations within WDR62 result in severe cerebral cortical malformations. One of the WDR62 mutant proteins found in a patient with microcephaly encodes a C-terminal truncated protein that fails to associate efficiently with JNK and MKK7β1. The present article shows that the WDR62 C-terminal region harbors a novel dimerization domain composed of a putative loop-helix domain that is necessary and sufficient for WDR62 dimerization and is critical for its scaffolding function. The loop-helix domain is highly conserved between orthologues and is also shared by the JNK scaffold protein, JNKBP1/MAPKBP1. Based on the high sequence conservation of the loop-helix domain, our article shows that MAPKBP1 homodimerizes and heterodimerizes with WDR62. Endogenous WDR62 and MAPKBP1 co-localize to stress granules following arsenite treatment, but not during mitosis. This study proposes another layer of complexity, in which coordinated activation of signaling pathways is mediated by the association between the different JNK scaffold proteins depending on their biological function.     

S100A14, a Member of the EF-hand Calcium-binding Proteins,Is Overexpressed in Breast Cancer and Acts as a Modulator of HER2 Signaling

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK. S100A14, one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens, and there is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding. Moreover, we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.