Adhesion G protein-coupled receptors (GPCR), with their very large and complex N termini, are thought to participate in cell-cell and cell-matrix interactions and appear to be highly relevant in several developmental processes. Their intracellular signaling is still poorly understood. Here we demonstrate that GPR133, a member of the adhesion GPCR subfamily, activates the G s protein/adenylyl cyclase pathway. The presence of the N terminus and the cleavage at the GPCR proteolysis site are not required for G protein signaling. G s protein coupling was verified by G␣ s knockdown with siRNA, overexpression of G␣ s , coexpression of the chimeric Gq s4 protein that routes GPR133 activity to the phospholipase C/inositol phosphate pathway, and missense mutation within the transmembrane domain that abolished receptor activity without changing cell surface expression. It is likely that not only GPR133 but also other adhesion GPCR signal via classical receptor/G protein-interaction.Adhesion receptors comprise the second largest subfamily of putatively G protein-coupled receptors (GPCR) 2 with more than 30 members in vertebrates (1, 2). Adhesion GPCR are characterized by long extracellular N termini, which are composed of multiple functional domains, a seven-transmembrane spanning (7TM) domain, and a cytoplasmic tail. Adhesion GPCR are believed to play a role in immune functions (3, 4), angiogenesis (5), cell polarity (6, 7), and development (8, 9). Mutations in some members of the protein family were identified as the cause of inherited developmental defects in humans such as Usher syndrome (VLGR1) (10) and bilateral frontoparietal polymicrogyria (GPR56) (11). Although there is consensus on the fact that this receptor class mediates essential cellcell and cell-matrix interactions (1, 12), the molecular mechanism of intracellular signal transduction of adhesion GPCR remains obscure.There are only a few studies on intracellular signaling mechanisms of adhesion GPCR. Latrophilin 1, the prototype of adhesion GPCR, induces intracellular Ca 2ϩ signaling upon interaction with the exogenous ligand ␣-latrotoxin (13, 14). GPR56 appears to activate the G 12/13 protein/Rho pathway after stimulation with an antibody against the ectodomain (15). BAI1 recognizes phosphatidylserine and can directly recruit a Racguanine nucleotide exchange factor (Rac-GEF) complex to mediate the uptake of apoptotic cells (16). The cytoplasmic domain of BAI2 interacts with GA-binding protein ␥, and GAbinding protein-␣/␥ or GA-binding protein-␣/␤ work as transcriptional repressors of VEGF (17). However, clear evidence of intracellular signaling for most adhesion GPCR via G proteins is still missing (12).Genetic variations in the GPR133 gene, also a member of the adhesion GPCR family, were associated with adult height (18) and the RR interval duration in electrocardiograms (19). GPR133 is expressed in CNS (20) and other tissues; its endogenous agonists and the signal transduction are unknown. Here we demonstrate that GPR133 is coupled to the G s protein/adeny...

The family of trace amine-associated receptors (TAAR) comprises 9 mammalian TAAR subtypes, with intact gene and pseudogene numbers differing considerably even between closely related species. To date the best characterized subtype is TAAR1, which activates the Gs protein/adenylyl cyclase pathway upon stimulation by trace amines and psychoactive substances like MDMA or LSD. Recently, chemosensory function involving recognition of volatile amines was proposed for murine TAAR3, TAAR4 and TAAR5. Humans can smell volatile amines despite carrying open reading frame (ORF) disruptions in TAAR3 and TAAR4. Therefore, we set out to study the functional and structural evolution of these genes with a special focus on primates. Functional analyses showed that ligands activating the murine TAAR3, TAAR4 and TAAR5 do not activate intact primate and mammalian orthologs, although they evolve under purifying selection and hence must be functional. We also find little evidence for positive selection that could explain the functional differences between mouse and other mammals. Our findings rather suggest that the previously identified volatile amine TAAR3–5 agonists reflect the high agonist promiscuity of TAAR, and that the ligands driving purifying selection of these TAAR in mouse and other mammals still await discovery. More generally, our study points out how analyses in an evolutionary context can help to interpret functional data generated in single species.

The blistering skin disorder Epidermolysis bullosa simplex (EBS) results from dominant mutations in K5 or K14 genes, encoding the intermediate filament network of basal epidermal keratinocytes. The mechanisms governing keratin network formation and collapse due to EBS mutations remain incompletely understood. Drosophila lacks cytoplasmic intermediate filaments, providing a ‚null’ environment to examine the formation of keratin networks and determine mechanisms by which mutant keratins cause pathology. Here, we report that ubiquitous co-expression of transgenes encoding wild-type human K14 and K5 resulted in the formation of extensive keratin networks in Drosophila epithelial and non-epithelial tissues, causing no overt phenotype. Similar to mammalian cells, treatment of transgenic fly tissues with phosphatase inhibitors caused keratin network collapse, validating Drosophila as a genetic model system to investigate keratin dynamics. Co-expression of K5 and a K14R125C mutant that causes the most severe form of EBS resulted in widespread formation of EBS-like cytoplasmic keratin aggregates in epithelial and non-epithelial fly tissues. Expression of K14R125C/K5 caused semi-lethality; adult survivors developed wing blisters and were flightless due to lack of intercellular adhesion during wing heart development. This Drosophila model of EBS is valuable for the identification of pathways altered by mutant keratins and for development of EBS therapies.

Chemokine receptors control leukocyte chemotaxis and cell-cell communication but have also been associated with pathogen entry. GPR33, an orphan member of the chemokine-like receptor family, is a pseudogene in most humans. After the appearance of GPR33 in first mammalian genomes, this receptor underwent independent pseudogenization in humans, other hominoids and some rodent species. It was speculated that a likely cause of GPR33 inactivation was its interplay with a rodenthominoid-specific pathogen. Simultaneous pseudogenization in several unrelated species within the last 1 million years (myr) caused by neutral drift appears to be very unlikely suggesting selection on the GPR33 null-allele. Although there are no signatures of recent selection on human GPR33 we found a significant increase in the pseudogene allele frequency in European populations when compared with African and Asian populations. Because its role in the immune system was still hypothetical expression analysis revealed that GPR33 is highly expressed in dendritic cells (DC). Murine GPR33 expression is regulated by the activity of toll-like receptors (TLR) and AP-1/NF-κB signaling pathways in cell culture and in vivo. Our data indicate an important role of GPR33 function ⋆ The sequences reported in this paper have been deposited in the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) database (Accession

No abstract