Constitutive endocytic cycle of the CB1 cannabinoid receptor

The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion ( approximately 85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.     

Brief communication: New Y‐chromosome binary markers improve phylogenetic resolution within haplogroup R1a1

Haplogroup R1a1‐M198 is a major clade of Y chromosomal haplogroups which is distributed all across Eurasia. To this date, many efforts have been made to identify large SNP‐based subgroups and migration patterns of this haplogroup. The origin and spread of R1a1 chromosomes in Eurasia has, however, remained unknown due to the lack of downstream SNPs within the R1a1 haplogroup. Since the discovery of R1a1‐M458, this is the first scientific attempt to divide haplogroup R1a1‐M198 into multiple SNP‐based sub‐haplogroups. We have genotyped 217 R1a1‐M198 samples from seven different population groups at M458, as well as the Z280 and Z93 SNPs recently identified from the “1000 Genomes Project”. The two additional binary markers present an effective tool because now more than 98% of the samples analyzed assign to one of the three sub‐haplogroups. R1a1‐M458 and R1a1‐Z280 were typical for the Hungarian population groups, whereas R1a1‐Z93 was typical for Malaysian Indians and the Hungarian Roma. Inner and Central Asia is an overlap zone for the R1a1‐Z280 and R1a1‐Z93 lineages. This pattern implies that an early differentiation zone of R1a1‐M198 conceivably occurred somewhere within the Eurasian Steppes or the Middle East and Caucasus region as they lie between South Asia and Eastern Europe. The detection of the Z93 paternal genetic imprint in the Hungarian Roma gene pool is consistent with South Asian ancestry and amends the view that H1a‐M82 is their only discernible paternal lineage of Indian heritage. © 2012 Wiley Periodicals, Inc.     

Microvascular dysfunction after transient high glucose is caused by superoxide-dependent reduction in the bioavailability of NO and BH(4)

We hypothesized that transient high-glucose concentration interferes with mediation by nitric oxide (NO) of flow-induced dilation (FID) of arterioles due to enhanced production of superoxide. In isolated, pressurized (80 mmHg) rat gracilis muscle arterioles ( approximately 130 microm) after transient high-glucose treatment (tHG; incubation with 30 mM glucose for 1 h), FID was reduced (maximum: control, 38 +/- 4%; after tHG, 17 +/- 3%), which was not further diminished by the NO synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME; 18 +/- 2%). Correspondingly, an enhanced polyethylene-glycol-SOD (PEG-SOD)-sensitive superoxide production was detected after tHG in carotid arteries by dihydroethydine (DHE) staining. Presence of PEG-SOD during tHG prevented the reduction of FID (41 +/- 3%), which could be inhibited by l-NAME (20 +/- 4%). Administration of PEG-SOD after tHG did not prevent the reduction of FID (22 +/- 3%). Sepiapterin, a precursor of the NO synthase cofactor tetrahydrobiopterin (BH(4)), administered during tHG did not prevent the reduction of FID (maximum, 15 +/- 5%); however, it restored FID when administered after tHG (32 +/- 4%). Furthermore, inhibition of either glycolysis by 2-deoxyglucose or mitochondrial complex II by 2-thenoyltrifluoroacetone reduced the tHG-induced DHE-detectable enhanced superoxide production in carotid arteries and prevented FID reduction in arterioles (39 +/- 5 and 35 +/- 2%). Collectively, these findings suggest that in skeletal muscle arterioles, a transient elevation of glucose via its increased metabolism, elicits enhanced production of superoxide, which decreases the bioavailability of NO and the level of the NOS cofactor BH(4), resulting in a reduction of FID mediated by NO.     

Common and specific properties of herpesvirus UL34/UL31 protein family members revealed by protein complementation assay

The proteins encoded by the UL34 and UL31 genes of herpes simplex virus are conserved among herpesviruses. They form a complex that is essential for the egress of the herpesvirus nucleocapsids from the nucleus. In previous work on the homologous protein complex in murine cytomegalovirus (MCMV), we defined their mutual binding domains. Here, we started to map binding domains within the UL34/UL31 proteins of alpha-, beta-, and gammaherpesviruses and to locate other functional properties. A protein complementation assay (PCA) using the TEM-1 beta-lactamase fragments fused to UL31 and UL34 protein homologues was used to study protein-protein interactions in cells. Wild-type MCMV M50 and M53 provided a strong reaction in the PCA, whereas mutants unable to form a complex did not. The homologous pairs of herpes simplex virus type 1, pseudorabies virus, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and murine herpes virus 68 proteins also reacted, with the exception of the EBV proteins. Cross-complementation was found to be positive only within the same herpesvirus subfamily. Moreover, the HCMV homologues rescued replication-defective MCMV genomes lacking one or the other gene. We identified the binding site of M53 for M50 in the first conserved region (CR1) (M. Loetzerich, Z. Ruzsics, and U. H. Koszinowski, J. Virol. 80:73-84). Here we show that the CR1 of all tested UL31 proteins contains the UL34 binding site, and chimeric proteins carrying the subfamily-specific CR1 rescued the ability to cross-complement in the PCA.     

MMP25 (MT6-MMP) is highly expressed in human colon cancer, promotes tumor growth, and exhibits unique biochemical properties

MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (K(i) = 0.2 nm) over TIMP-2 (K(i) = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.     

Estimation of the number of angiotensin II AT1 receptors in rat kidney afferent and efferent arterioles

OBJECTIVE: To examine the effects of the renin-angiotensin system (RAS) on renal arterioles to determine the association between the distribution of angiotensin II AT1 receptors and the morphologic and physiologic heterogeneity of renal arterioles. STUDY DESIGN: To estimate the number of angiotensin II AT1 receptors along the length of the arterioles and per arteriole, we combined immunoelectron microscopy with stereology. RESULTS: The number of AT1 receptor molecules was significantly lower in the renin-positive smooth muscle cells (SMCs) than in the renin-negative SMCs of the afferent and efferent arterioles. There were no significant differences along and between the afferent and efferent arterioles in relative number of AT1 receptors of endothelial cells or SMCs. CONCLUSION: Our results suggest that the heterogeneous activity of angiotensin II in SMCs and the different permeabilities of the endothelium along the afferent arterioles are probably not controlled directly by angiotensin II AT1 receptors. However, the activity of the RAS is possibly involved in the significantly reduced number of receptors in renin-granulated cells. An understanding of how the number of AT1 receptors on the SMC surface is controlled may furnish a new path for pharmacologically changing RAS activity on SMCs.     

Mutation in a gene required for lipopolysaccharide and enterobacterial common antigen biosynthesis affects virulence in the plant pathogen Erwinia carotovora subsp. atroseptica.

Spontaneous bacteriophage-resistant mutants of the phytopathogen Erwinia carotovora subsp. atroseptica (Eca) SCRI1043 were isolated and, out of 40, two were found to exhibit reduced virulence in planta. One of these mutants, A5/22, showed multiple cell surface defects including alterations in synthesis of outer membrane proteins, lipopolysaccharide (LPS), enterobacterial common antigen (ECA), and flagella. Mutant A5/22 also showed reduced synthesis of the exoenzymes pectate lyase (Pel) and cellulase (Cel), major virulence factors for this pathogen. Genetic analysis revealed the pronounced pleiotropic mutant phenotype to be due to a defect in a single gene (rffG) that, in Escherichia coli, is involved in the production of ECA. We also show that while other enteric bacteria possess duplicate homologues of this gene dedicated separately to synthesis of LPS and ECA, Eca has a single gene.     

The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptor-stimulated cannabinoid CB1 receptor activity

The cannabinoid CB1 receptor (CB1R) is a G protein-coupled receptor, which couples to the Gi/o family of heterotrimeric G proteins. The receptor displays both basal and agonist-induced signaling and internalization. Although basal activity of CB1Rs is attributed to constitutive (agonist-independent) receptor activity, studies in neurons suggested a role of postsynaptic endocannabinoid (eCB) release in the persistent activity of presynaptic CB1Rs. To elucidate the role of eCBs in basal CB1R activity, we have investigated the role of diacylglycerol lipase (DAGL) in this process in Chinese hamster ovary (CHO) cells, which are not targeted specifically with eCBs. Agonist-induced G protein activation was determined by detecting dissociation G protein subunits expressed in CHO cells with bioluminescence resonance energy transfer (BRET), after labeling the alpha and beta subunits with Renilla luciferase and enhanced yellow fluorescent protein (EYFP), respectively. Preincubation of the cells with tetrahydrolipstatin (THL), a known inhibitor of DAGLs, caused inhibition of the basal activity of CB1R. Moreover, preincubation of CHO and cultured hippocampal neurons with THL increased the number of CB1Rs on the cell membrane, which reflects its inhibitory action on CB1R internalization in non-simulated cells. In CHO cells co-expressing CB1R and angiotensin AT1 receptors, angiotensin II-induced Go protein activation that was blocked by both a CB1R antagonist and THL. These data indicate that cell-derived eCB mediators have a general role in the basal activity of CB1Rs in non-neural cells and neurons, and that this mechanism can be stimulated by AT1 receptor activation.