The type 2 dopamine receptor D₂ (D₂-R), member of the G protein-coupled receptor (GPCR) superfamily, exists in two isoforms, short (D_2S-R) and long (D_2L-R). They differ by an additional 29 amino acids (AA) in the third cytoplasmic loop (ICL3) of the D_2L-R. These isoforms differ in their intracellular localization and trafficking functionality, as D_2L-R possesses a larger intracellular pool, mostly in the endoplasmic reticulum (ER). This review focuses on the evolutionarily conserved motifs in the ICL3 of the D₂-R and proteins interacting with the ICL3 of both isoforms, specifically with the 29 AA insert. These motifs might be involved in D₂-R exit from the ER and have an impact on cell-surface and intracellular localization and, therefore, also play a role in the function of dopamine receptor signaling, ligand binding and possible homo/heterodimerization. Our recent bioinformatic data on potential new interaction partners for the ICL3 of D₂-Rs are also presented. Both are highly relevant, and have clinical impacts on the pathophysiology of several diseases such as Parkinson’s disease, schizophrenia, Tourette’s syndrome, Huntington’s disease, manic depression, and others, as they are connected to a variety of essential motifs and differences in communication with interaction partners.

Background: 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.Methods: A novel real-time fluorescence resonance energy transfer (FRET)-based internalization assay combined with dominant-negative variants of dynamin-1 (Dyn K44A) and the chemical clathrin inhibitor Pitstop2 in HEK-293A cells was used to study the effect of specific endocytic proteins on BILF1 internalization. Bioluminescence resonance energy transfer (BRET)-saturation analysis was used to study BILF1 receptor interaction with β-arrestin2 and Rab7. In addition, a bioinformatics approach informational spectrum method (ISM) was used to investigate the interaction affinity of BILF1 receptors with β-arrestin2, AP-2, and caveolin-1.Results: We identified dynamin-dependent, clathrin-mediated constitutive endocytosis for all BILF1 receptors. The observed interaction affinity between BILF1 receptors and caveolin-1 and the decreased internalization in the presence of a dominant-negative variant of caveolin-1 (Cav S80E) indicated the involvement of caveolin-1 in BILF1 trafficking. Furthermore, after BILF1 internalization from the plasma membrane, both the recycling and degradation pathways are proposed for BILF1 receptors.Conclusions: The similarity in the internalization mechanisms observed for EBV-BILF1 and PLHV1-2 BILF1 provide a foundation for further studies exploring a possible translational potential for PLHVs, as proposed previously, and provides new information about receptor trafficking.Graphical

Extensive ligand-receptor promiscuity in the chemokine signaling system balances beneficial redundancy and specificity. However, this feature poses a major challenge to selectively modulate the system pharmacologically. Here, we identified a conserved cluster of three aromatic receptor residues that anchors the second extracellular loop (ECL2) to the top of receptor transmembrane helices (TM) 4 and 5 and enables recognition of both shared and specific characteristics of interacting chemokines. This cluster was essential for the activation of several chemokine receptors. Furthermore, characteristic motifs of the ß ₁ strand and 30s loop make the two main CC-chemokine subgroups—the macrophage inflammatory proteins (MIPs) and monocyte chemoattractant proteins (MCPs)—differentially dependent on this cluster in the promiscuous receptors CCR1, CCR2, and CCR5. The cluster additionally enabled CCR1 and CCR5 to discriminate between closely related MIPs based on the N terminus of the chemokine. G protein signaling and β-arrestin2 recruitment assays confirmed the importance of the conserved cluster in receptor discrimination of chemokine ligands. This extracellular site may facilitate the development of chemokine-related therapeutics.

The ingestion of hydrolysable tannins as a potential nutrient to reduce boar odor in entire males results in the significant enlargement of parotid glands (parotidomegaly). The objective of this study was to characterize the effects of different levels of hydrolysable tannins in the diet of fattening boars (n = 24) on salivary gland morphology and proline-rich protein (PRP) expression at the histological level. Four treatment groups of pigs (n = 6 per group) were fed either a control (T0) or experimental diet, where the T0 diet was supplemented with 1% (T1), 2% (T2), or 3% (T3) of the hydrolysable tannin-rich extract Farmatan^®. After slaughter, the parotid and mandibular glands of the experimental pigs were harvested and dissected for staining using Goldner’s Trichrome method, and immunohistochemical studies with antibodies against PRPs. Morphometric analysis was performed on microtome sections of both salivary glands, to measure the acinar area, the lobular area, the area of the secretory ductal cells, and the sizes of glandular cells and their nuclei. Histological assessment revealed that significant parotidomegaly was only present in the T3 group, based on the presence of larger glandular lobules, acinar areas, and their higher nucleus to cytoplasm ratio. The immunohistochemical method, supported by color intensity measurements, indicated significant increases in basic PRPs (PRB2) in the T3 and acidic PRPs (PRH1/2) in the T1 groups. Tannin supplementation did not affect the histo-morphological properties of the mandibular gland. This study confirms that pigs can adapt to a tannin-rich diet by making structural changes in their parotid salivary gland, indicating its higher functional activity.

Endocytosis is a fundamental process involved in trafficking of various extracellular and transmembrane molecules from the cell surface to its interior. This enables cells to communicate and respond to external environments, maintain cellular homeostasis, and transduce signals. G protein-coupled receptors (GPCRs) constitute a family of receptors with seven transmembrane alpha-helical domains (7TM receptors) expressed at the cell surface, where they regulate physiological and pathological cellular processes. Several herpesviruses encode receptors (vGPCRs) which benefits the virus by avoiding host immune surveillance, supporting viral dissemination, and thereby establishing widespread and lifelong infection, processes where receptor signaling and/or endocytosis seem central. vGPCRs are rising as potential drug targets as exemplified by the cytomegalovirus-encoded receptor US28, where its constitutive internalization has been exploited for selective drug delivery in virus infected cells. Therefore, studying GPCR trafficking is of great importance. This review provides an overview of the current knowledge of endocytic and cell localization properties of vGPCRs and methodological approaches used for studying receptor internalization. Using such novel approaches, we show constitutive internalization of the BILF1 receptor from human and porcine γ-1 herpesviruses and present motifs from the eukaryotic linear motif (ELM) resources with importance for vGPCR endocytosis.