Corrosion cast study of the canine hepatic veins

This study presents a detailed description of the distribution, diameters and drainage patterns of hepatic veins on the basis of the corrosion cast analysis in 18 dogs. We classified the hepatic veins in three main groups: the right hepatic veins of the caudate process and right lateral liver lobe, the middle hepatic veins of the right medial and quadrate lobes and the left hepatic veins of both left liver lobes and the papillary process. The corrosion cast study showed that the number of the veins in the Nomina Anatomica Veterinaria and most anatomical textbooks is underestimated. The number of various-sized hepatic veins of the right liver division ranged from 3 to 5 and included 1 to 4 veins from the caudate process and 2 to 4 veins from the right lateral liver lobe. Generally, in all corrosion casts, one middle-sized vein from the right part of the right medial lobe, which emptied separately in the caudal vena cava, was established. The other vein was a large-sized vein from the remainder of the central division, which frequently joined the common left hepatic vein from the left liver lobes. The common left hepatic vein was the largest of all the aforementioned hepatic veins.

Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis

The viral G-protein-coupled receptor (vGPCR) BILF1 encoded by the Epstein–Barr virus (EBV) is an oncogene and immunoevasin and can downregulate MHC-I molecules at the surface of infected cells. MHC-I downregulation, which presumably occurs through co-internalization with EBV-BILF1, is preserved among BILF1 receptors, including the three BILF1 orthologs encoded by porcine lymphotropic herpesviruses (PLHV BILFs). This study aimed to understand the detailed mechanisms of BILF1 receptor constitutive internalization, to explore the translational potential of PLHV BILFs compared with EBV-BILF1

Regulation of GIP and GLP1 Receptor Cell Surface Expression by N-Glycosylation and Receptor Heteromerization

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer

Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET2

Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET2 pair consisting of the fusion proteins CK2 alpha-Rluc8 and CK2 alpha-GFP(2). This BRET2 sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET2 sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (G(alpha s)) and the polycationic compound polylysine. G(alpha s), but not the CK2 substrate beta-arrestin2, decreased the BRET2 signal by up to 50 %. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50 %. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET2 sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors

Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to β-arrestin-dependent internalization

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a beta -arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize beta -arrestin, Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta -arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not beta -arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail, We thus investigated a role for CKII sites in determining GPCR internalization via beta -arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta -arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A/T371A,S383A) in the C-tail of a p-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta -arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta -arrestin-dependence was observed. Visualization of beta -arrestin/GFP redistribution confirmed a loss or gain of beta -arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta -arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for beta -arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by beta -arrestin, thus providing further support for the involvement of CKII, This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the beta -arrestin-dependent pathway