Protein expression of KIT, BRAF, GNA11, GNAQ and RASSF1 in feline diffuse iris melanomas.

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/µm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.

Gαq controls rheumatoid arthritis via regulation of Th17 differentiation.

Gαq, the α-subunit of Gq protein, is ubiquitously expressed in mammalian cells. It initially attracted attention for its physiological significance in cardiovascular system. In recent years, studies have also indicated the important roles of Gαq in regulating immunity, supplying us a new insight into the mechanism of immune regulation. T helper type 17 (Th17) cells are potent inducers of tissue inflammation. Many studies have shown that Th17 cells are major effector cells in the pathogenesis of many experimental autoimmune diseases and human inflammatory conditions such as rheumatoid arthritis (RA). One of our previous studies has shown that Gαq negatively controls the disease activity of RA. However, how Gαq controls the pathogenesis of autoimmune disease is not clear. Whether this effect is via the regulation of Th17 differentiation is still not known. We aimed to find out the role of Gαq in control of Th17 differentiation. We investigated the relationship between Gαq and Th17 in RA patients. We then investigated the mechanism of how Gαq regulated Th17 differentiation by using Gnaq(-/-) mice. We observed that the expression of Gαq was negatively associated with interleukin-17A expression in RA patients, indicating that Gαq negatively controlled the differentiation of Th17 cells. By using Gnaq(-/-) mice, we demonstrated that Gαq inhibited the differentiation of Th17 cell via regulating the activity of extracellular signal-regulated kinase-1/2 to control the expression of STAT3 (signal transducer and activator of transcription 3) and RORα (RAR-related orphan receptor-α). These data suggest the possibility of targeting Gαq to develop a novel therapeutic regimen for autoimmune disease.

Tissue distribution and functional analyses of the constitutively active orphan G protein coupled receptors, GPR26 and GPR78.

GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.

Molecular characterization of a G protein alpha-subunit-encoding gene from Mucor circinelloides.

Genes encoding the Galpha subunit were cloned from Mucor circinelloides, a zygomycete dimorphic fungus. There are at least four genes that encode for Galpha subunits, gpa1, gpa2, gpa3, and gpa4. The genes gpa1 and gpa3 were isolated and characterized, and their predicted products showed 36%-67% identity with Galpha subunits from diverse fungi. Northern blot analysis of gpa3 showed that it is present in spores and constitutively expressed during mycelium development and during yeast-mycelium and mycelium-yeast transitions. However, during yeast cell growth, decreased levels of mRNA were observed. Sequence analysis of gpa3 cDNA revealed that Gpa3 encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40.8 kDa. The deduced sequence of Gpa3 protein contains all the consensus regions of Galpha subunits of the Galpha(i/o/t) subfamily except the cysteine near the C terminus for potential ADP-ribosylation by pertussis toxin. This cDNA was expressed in Escherichia coli and purified by affinity chromatography. Based on its electrophoretic mobility in SDS-PAGE, the molecular mass of the His6-tagged Gpa3 was 45 kDa. The recombinant protein was recognized by a polyclonal antibody against a fragment of a human Galpha(i/o/t). Furthermore, the recombinant Gpa3 was ADP-ribosylated by activated cholera toxin and [32P]NAD but not by pertussis toxin. These results indicate that in M. circinelloides the Galpha subunit Gpa3 is expressed constitutively during differentiation.

Genetics and proteomics of pituitary tumors.

Genetics and proteomics determine structure and function of normal tissues, and the molecular alterations that underlie tumorigenesis result in changes in these aspects of tissue biology in neoplasms. We review the known genetic alterations in pituitary tumors. These include the oncogenic Gsalpha protein (GSP)-activating mutations, and pituitary tumor-derived fibroblast growth factor receptor-4 (ptd-FGFR4), as well as tumor suppressor gene mutations associated with multiple endocrine neoplasia type 1 (MEN1). Other candidates identified from expression profiling include pituitary tumor-transforming gene (PTTG), GADD45, and bone morphogenic protein (BMP)4. Proteomic changes in pituitary tumors include classical alterations identified by immunohistochemistry as well as epigenetic reductions in p27. The underlying mechanisms for dysregulated cell adhesive molecules including cadherins and FGFRs are reviewed. The combined use of genetic and proteomic approaches will enhance novel drug therapeutic development.

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling.

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.