Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

Calcitonin gene‐related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor‐like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP‐CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1‐CLR ECD fusion purified as a monomer, whereas the RAMP2‐CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27‐37) and AM(37‐52) fragments were identified as the minimal ECD complex binding regions. The CGRP C‐terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C‐terminal amide group was essential for ECD binding. Alanine‐scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.     

Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor

The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production.     

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for ¹³C labeling of the C-terminal domain of angiopoietin-2, ¹⁵N labeling of ubiquitin and for ²H/¹³C/¹⁵N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.     

Study of protein-protein interactions by fluorescence of tryptophan analogs: application to immunoglobulin G binding domain of streptococcal protein G

The protein-protein interaction system often contains many fluorophores that may significantly interfere with the quantitative determination of the binding abilities. To solve this perplexing problem, we biosynthetically incorporated the two tryptophan analogs, 5-hydroxytryptophan and 7-azatryptophan, into the immunoglobulin G (IgG) binding domain of streptococcal protein G (PGBD). The exclusive excitation and novel fluorescence changes in both the intensity and anisotropy are beneficial to reporting the details of the interactions between PGBD and the IgG fragments and enable assessment of the binding abilities. The dissociation constants are estimated to be 0.28 microM for the binding of human Fc and 8.0 microM for mouse Fc. The results clearly demonstrate that labeling of tryptophan analogs has very little effect on the binding abilities and is broadly applicable to quantitatively studying protein-protein interactions in a whole biomolecular complex.     

Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins

P2Y receptors are G protein coupled receptors that respond to extracellular nucleotides to promote a multitude of signaling events. Our laboratory has purified several P2Y receptors with the goal of providing molecular insight into their: (1) ligand binding properties, (2) G protein signaling selectivities, and (3) regulation by RGS proteins and other signaling cohorts. The human P2Y₁ receptor and the human P2Y₁₂ receptor, both of which are intimately involved in ADP-mediated platelet aggregation, were purified to near homogeneity and studied in detail. After high-level expression from recombinant baculovirus infection of Sf9 insect cells, approximately 50% of the receptors were successfully extracted with digitonin. Purification of nearly homogeneous epitope-tagged P2Y receptor was achieved using metal-affinity chromatography followed by other traditional chromatographic steps. Yields of purified P2Y receptors range from 10 to 100 g/l of infected cells. Once purified, the receptors were reconstituted in model lipid vesicles along with their cognate G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase assays demonstrated the functional activity of the reconstituted purified receptor. We have utilized this reconstitution system to assess the action of various nucleotide agonists and antagonists, the relative G protein selectivity, and the influence of other proteins, such as phospholipase C, on P2Y receptor-promoted signaling. Furthermore, we have identified the RGS expression profile of platelets and have begun to assess the action of these RGS proteins in a reconstituted P2Y receptor/G protein platelet model.     

D(2)-Dopamine receptors target regulator of G protein signaling 9-2 to detergent-resistant membrane fractions

Detergent-resistant membranes (DRM) are thought to contain structures such as lipid rafts that are involved in compartmentalizing cell membranes. We report that the majority of D(2)-dopamine receptors (D(2)R) expressed endogenously in mouse striatum or expressed in immortalized cell-lines is found in DRM. In addition, exogenous co-expression of D(2)R in a cell line shifted the expression of regulator of G protein signaling 9-2 (RGS9-2) into DRM. RGS9-2 is a protein that is highly enriched in the striatum and specifically regulates striatal D(2)R. In the striatum, RGS9-2 is mostly associated with DRMs but when expressed in cell lines, RGS9-2 is present in the soluble cytoplasmic fraction. In contrast, the majority of mu opioid receptors and delta opioid receptors are found in detergent-soluble membrane and there was no shift of RGS9-2 into DRM after co-expression of mu opioid receptor. These data suggest that the targeting of RGS9-2 to DRM in the striatum is mediated by D(2)R and that DRM is involved in the formation of a D(2)R signaling complex. D(2)R-mediated targeting of RGS9-2 to DRM was blocked by the deletion of the RGS9-2 DEP domain or by a point mutation that abolishes the GTPase accelerating protein function of RGS9-2.     

G-protein-coupled receptor domain overexpression in Halobacterium salinarum: long-range transmembrane interactions in heptahelical membrane proteins

The aminergic alpha(2b)-adrenergic receptor (alpha(2b)-AR) third intracellular loop (alpha(2b)-AR 3i) mediates receptor subcellular compartmentalization and signal transduction processes via ligand-dependent interaction with G(i)- and G(o)- proteins. To understand the structural origins of these processes we engineered several lengths of alpha(2b)-AR 3i into the third intracellular loop of the proton pump bacteriorhodopsin (bR) and produced the fusion proteins in quantities suitable for physical studies. The fusion proteins were expressed in the Archaeon Halobacterium salinarum and purified. A highly expressed fusion protein was crystallized from bicelles and diffracted to low resolution on an in-house diffractometer. The bR-alpha(2b)-AR 3i(203-292) protein possessed a photocycle slightly perturbed from that of the wild-type bR. The first half of the fusion protein photocycle, correlated with proton release, is accelerated by a factor of 3, whereas the second half, correlated with proton uptake, is slightly slower than wild-type bR. In addition, there is a large decrease in the pK(a), (from 9.6 to 8.3) of the terminal proton release group in the unphotolyzed state of bR-alpha(2b)-AR 3i as deduced from the pH-dependence of the M-formation. Perturbation of a cytoplasmic loop has thus resulted in the perturbation of proton release at the extracellular surface. The current work indicates that long-range and highly coupled intramolecular interactions exist that are capable of "transducing" structural perturbations (e.g., signals) across the cellular membrane. This gene fusion approach may have general applicability for physical studies of G-protein-coupled receptor domains in the context of the bR structural scaffold.     

Biosynthesis of peptide fragments of eukaryotic GPCRs in Escherichia coli by directing expression into inclusion bodies

Biosynthesis of peptides in heterologous systems is often a prerequisite to biophysical analyses. Large amounts of peptides, in particular portions of membrane proteins, are needed to optimize conditions for secondary and tertiary structure analysis. Chemical synthesis of these peptides is limited by their high hydrophobicity and also due to the need to incorporate isotopic labels for high resolution NMR analysis. In this protocol, we describe a method for the heterologous expression and purification of unlabeled and isotopically labeled peptide fragments of Ste2p, an integral membrane G protein‐coupled receptor. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

The NMR-derived conformation of neuropeptide AF,an orphan G-protein coupled receptor peptide

The tertiary structure of the pain modulating and anti-opiate neuropeptide, human neuropeptide AF (NPAF) (the sequence is AGEGLNSQFWSLAAPQRF-NH(2)), was determined by (1)H-NMR. The structure of NPAF was determined in two solvent systems, namely 50%/50% trifluoroethanol-d(3)/H(2)O (TFE/H(2)O) and in the cell membrane mimetic micelle, sodium dodecylsulfate-d(25) (SDS). The receptor for NPAF is an orphan G-protein coupled receptor, and the micellar SDS solvent system was used to emulate the cell membrane surface in line with the Cell Membrane Compartments Theory proposed by R. Schwyzer (Biopolymers, 1995, Vol. 37, pp. 5-16). In both solvent systems, NPAF was found to be primarily alpha-helical within the central portion of the molecule, from Asn(6) to Ala(14). The N-terminus was random in both solvent systems. In the SDS solution, the C-terminal tetrapeptide was structured and formed a type I beta-turn, whereas in TFE/H(2)O it was unstructured, showing the importance of the C-terminal tetrapeptide in receptor recognition. NPAF was found to associate with SDS, and was shown to be near the surface of the micelle by spin label studies with 5-doxyl-stearic acid.     

Mechanisms of the anterograde trafficking of GPCRs: Regulation of AT1R transport by interacting proteins and motifs

Anterograde cell surface transport of nascent G protein‐coupled receptors (GPCRs) en route from the endoplasmic reticulum (ER) through the Golgi apparatus represents a crucial checkpoint to control the amount of the receptors at the functional destination and the strength of receptor activation‐elicited cellular responses. However, as compared with extensively studied internalization and recycling processes, the molecular mechanisms of cell surface trafficking of GPCRs are relatively less defined. Here, we will review the current advances in understanding the ER‐Golgi‐cell surface transport of GPCRs and use angiotensin II type 1 receptor as a representative GPCR to discuss emerging roles of receptor‐interacting proteins and specific motifs embedded within the receptors in controlling the forward traffic of GPCRs along the biosynthetic pathway.      

Global fold of human cannabinoid type 2 receptor probed by solid‐state 13C‐, 15N‐MAS NMR and molecular dynamics simulations

The global fold of human cannabinoid type 2 (CB₂) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state ¹³C‐ and ¹⁵N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly ¹³C‐ and ¹⁵N‐labeled CB₂ receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. ¹³C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of C_α, C_β, and C?O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the ¹³C chemical shift distribution of C_α resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB₂. Thus the shape of the C_α band can be used as an indicator of CB₂ global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional ¹³C_α(i)? ¹³C?O(i) and ¹³C?O(i)? ¹⁵NH(i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB₂ by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc.     

New approaches towards the understanding of integral membrane proteins: A structural perspective on G protein‐coupled receptors

Three‐dimensional structure determination of integral membrane proteins has advanced in unprecedented detail our understanding of mechanistic events of how ion channels, transporters, receptors, and enzymes function. This exciting progress required a tremendous amount of methods development, as exemplified here with G protein‐coupled receptors (GPCRs): Optimizing the production of GPCRs in recombinant hosts; increasing the probability of crystal formation using high‐affinity ligands, nanobodies, and minimal G proteins for co‐crystallization, thus stabilizing receptors into one conformation; using the T4 lysozyme technology and other fusion partners to promote crystal contacts; advancing crystallization methods including the development of novel detergents, and miniaturization and automation of the lipidic cubic phase crystallization method; the concept of conformational thermostabilization of GPCRs; and developing microfocus X‐ray synchrotron technologies to analyze small GPCR crystals. However, despite immense progress to explain how GPCRs function, many receptors pose intractable hurdles to structure determination at this time. Three emerging methods, serial femtosecond crystallography, micro electron diffraction, and single particle electron cryo‐microscopy, hold promise to overcome current limitations in structural membrane biology.     

Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide-reactive protein of the adenosinetriphosphatase complex of escherichia coli

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1-butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N-methylpyrrolidone. At relatively low concentrations (< 3 M) aprotic solvents inhibited β-D-galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport-positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport-negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D-lactate) or by a K⁺ diffusion potential established with the aid of valinomycin. The dicyclohexylcarbodiimide (DCCD)-reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE-cellulose and hydroxypropyl-Sephadex G-50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000. The purified and unlabeled DCCD-reactive protein was incorporated into K⁺-loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD-reactive protein alone made the membrane specifically permeable for protons.     

V1b vasopressin receptor trafficking and signaling: Role of arrestins,G proteins and Src kinase

The signaling pathway of G protein‐coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V_1b subtype (V_1bR) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of β‐arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V_1bR‐mediated MAP kinase pathway. Using MEF cells Knocked‐out for β‐arrestins 1 and 2, we demonstrated that both β‐arrestins 1 and 2 play a fundamental role in internalization and recycling of V_1bR with a rapid and transient V_1bR‐β‐arrestin interaction in contrast to a slow and long‐lasting β‐arrestin recruitment of the V₂ vasopressin receptor subtype (V₂R). Using V_1bR‐V₂R chimeras and V_1bR C‐terminus truncations, we demonstrated the critical role of the V_1bR C‐terminus in its interaction with β‐arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation‐independent manner. In parallel, V_1bR MAP kinase activation was dependent on arrestins and Src‐kinase but independent on G proteins. Interestingly, Src interacted with hV_1bR at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V_1bR involving both arrestins and Src kinase family.      

Comparison by photoaffinity labeling of the proteins involved in GTP hydrolysis from 70S ribosomes and a 5S RNA-protein complex from Bacillus stearothermophilus.

Photoaffinity labeling of 70S ribosomes from B. stearothermophilus by [³H]-1-(4-azidophenyl)-2-(5′-guanyl) pyrophosphate (APh-GDP) in the presence of fusidate and elongation factor G (EF-G) results in incorporation of tritium in the 50S proteins BL2, BL10 and BL22. Irradiation of the corresponding 5S RNA-protein complex in the presence of the GDP derivative gives only incorporation of tritium in BL10 and BL22. The proteins BL10 and BL22 comigrate in two dimensional gel electrophoresis with the 50S ribosomal proteins EL11 and EL18 from E. coli. The result suggests that the region at or near the guanine nucleotide binding site of the ribosome and the complex are the same. Since previous work has shown that the latter two are labeled upon irradiation of the ribosome with [³H]-APh-GDP, it is concluded that ribosomes from E. coli and B. stearothermophilus have structurally related GTPase sites.