Debra A. Kendall

Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides.

In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins. In this study we use cross‐linking to demonstrate that E. coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins. Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo. A second E. coli component, which is identified as trigger factor, can be efficiently cross‐linked to all tested nascent chains derived from both secreted and cytosolic proteins. We propose that SRP and trigger factor act as secretion‐specific and general molecular chaperone respectively, early in protein synthesis.

Signal peptides: exquisitely designed transport promoters

Prokaryotic proteins destined for transport out of the cytoplasm typically contain an N‐terminal extension sequence, called the signal peptide, which is required for export, it is evident that many secretory proteins utilize a common export system, yet the signal sequences themselves display very little primary sequence homology. in attempting to understand how different signal peptides are able to promote protein secretion through the same pathway, the physical features of natural signal sequences have been extensively examined for similarities that might play a part in function. Experimental data have confirmed statistical analyses which highlighted dominant features of natural signal sequences in Escherichia coli: a net positive charge in the N‐terminus increases efficiency of transport; the core region must maintain a threshold level of hydrophoblcity within a range of length limitations; the central portion adopts an α‐hellcal conformation in hydrophobic environments; and the signal cleavage region is ideally six residues long, with small side‐chain amino acids in the −1 and −3 positions. This review focuses on the parallels between signal peptide physical features and their functions, which emerge when the results of a variety of experimental approaches are combined. The requirement for each property may be ascribed to a potential interaction that is critical for efficient protein export. The summation of the key physical features produces signal peptides with the flexibility to function in multiple roles in order to expedite secretion. In this way, nature has indeed evolved exquisitely tuned signal sequences.

Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor

The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross‐linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross‐linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.

A simple procedure for the determination of the trapped volume of liposomes

Abstract A new method is described for determining the volume of the aqueous compartment of liposomes. Liposomes are prepared in a solution of the fluorescent dye, calcein. The fraction of the total volume that is within the liposomes is obtained as the fraction of the fluorescence that remains after adding cobalt(II) ions which, when chelated by calcein, quench its fluorescence. The method is rapid, simple and accurate. Separation of the liposomes from the medium is not required. The procedure is equally well suited to the assay of permeability characteristics of liposomal membranes.

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes.

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturing them. Various concentrations each of detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents most effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.

Oleamide is a selective endogenous agonist of rat and human CB1 cannabinoid receptors

The ability of the endogenous fatty acid amide, cis‐oleamide (ODA), to bind to and activate cannabinoid CB1 and CB2 receptors was investigated. ODA competitively inhibited binding of the nonselective cannabinoid agonist [3H]CP55,940 and the selective CB1 antagonist [3H]SR141716A to rat whole‐brain membranes with Ki values of 1.14 μM (0.52–2.53 μM, Hill slope=0.80, n=6) and 2.63 μM (0.62–11.20 μM, Hill slope=0.92, n=4), respectively. AEA inhibited [3H]CP55,940 binding in rat whole‐brain membranes with a Ki of 428 nM (346–510 nM, Hill slope=−1.33, n=3). ODA competitively inhibited [3H]CP55,940 binding in human CB1 (hCB1) cell membranes with a Ki value of 8.13 μM (4.97–13.32 μM, n=2). In human CB2 transfected (hCB2) HEK‐293T cell membranes, 100 μM ODA produced only a partial (42.5±7%) inhibition of [3H]CP55,940 binding. ODA stimulated [35S]GTPγS binding in a concentration‐dependent manner (EC50=1.64 μM (0.29–9.32 μM), R2=0.99, n=4–9), with maximal stimulation of 188±9% of basal at 100 μM. AEA stimulated [35S]GTPγS binding with an EC50 of 10.43 μM (4.45–24.42 μM, R2=1.00, n=3, 195±4% of basal at 300 μM). Trans‐oleamide (trans‐ODA) failed to significantly stimulate [35S]GTPγS binding at concentrations up to 100 μM. ODA (10 μM)‐stimulated [35S]GTPγS binding was reversed by the selective CB1 antagonist SR141716A (IC50=2.11 nM (0.32–13.77 nM), R2=1.00, n=6). The anatomical distribution of ODA‐stimulated [35S]GTPγS binding in rat brain sections was indistinguishable from that of HU210. Increases of similar magnitude were observed due to both agonists in the striatum, cortex, hippocampus and cerebellum. ODA (10 μM) significantly inhibited forskolin‐stimulated cyclic AMP (cAMP) accumulation in mouse neuroblastoma N1E 115 cells (P=0.02, n=11). ODA‐mediated inhibition was completely reversed by 1 μM SR141716A (P<0.001, n=11) and was also reversed by pretreatment with 300 ng ml−1 pertussis toxin (P<0.001, n=6). These data demonstrate that ODA is a full cannabinoid CB1 receptor agonist. Therefore, in addition to allosteric modulation of other receptors and possible entourage effects due to fatty acid amide hydrolase inhibition, the effects of ODA may be mediated directly via the CB1 receptor.

Involvement of the carboxyl terminus of the third intracellular loop of the cannabinoid CB1 receptor in constitutive activation of Gs.

Abstract: The human cannabinoid receptor CB1 functionally couples primarily to Gi‐, but also to Gs‐mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl‐terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist‐independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs‐ and not a Gi‐mediated pathway. Treatment with the CB1‐specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP‐55,940 to the L341A/A342L receptor was not markedly different from that for the wild‐type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.

Ligand binding and modulation of cyclic AMP levels depend on the chemical nature of residue 192 of the human cannabinoid receptor 1.

Abstract: The human cannabinoid receptor associated with the CNS (CB1) binds Δ9‐tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein‐coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys192 of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild‐type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP‐55,940 and the aminoalkylindole WIN 55,212‐2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP‐55,940, whereas WIN 55,212‐2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand‐induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC50 value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212‐2, an order of magnitude difference was observed for the EC50 for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP‐55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212‐2 binding, the data suggest a role for Lys192 in WIN 55,212‐2‐induced receptor activation.

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14.

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.

Distinct Roles of β-Arrestin 1 and β-Arrestin 2 in ORG27569-induced Biased Signaling and Internalization of the Cannabinoid Receptor 1 (CB1)

Background: CB1 is activated by agonist CP55940 in a G protein-dependent manner. Results: β-Arrestin 2 plays a role in CB1 internalization, whereas β-arrestin 1 is critical for ORG27569-induced ERK1/2, MEK1/2, and c-Src phosphorylation. Conclusion: Allosteric modulator ORG27569 endows CB1 with downstream signaling selectivity. Significance: This work discusses the first case of β-arrestin involvement in CB1-biased signaling. The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy in HEK293 cells and rat brain expressing the CB1 receptor (Ahn, K. H., Mahmoud, M. M., and Kendall, D. A. (2012) J. Biol. Chem. 287, 12070–12082). Here, we identify the mediators of CB1 receptor internalization and ORG27569-induced G protein-independent signaling. Using siRNA technology, we elucidate an ORG27569-induced signaling mechanism for CB1 wherein β-arrestin 1 mediates short term signaling to ERK1/2 with a peak at 5 min and other upstream kinase components including MEK1/2 and c-Src. Consistent with these findings, we demonstrate co-localization of CB1-GFP with red fluorescent protein-β-arrestin 1 upon ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of β-arrestin 2 in CB1 receptor internalization upon treatment with CP55940 (agonist) or treatment with ORG27569. These results demonstrate for the first time the involvement of β-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two β-arrestin isoforms in CB1 signaling and internalization.