Deconstructing G Protein-Coupled Receptor Dimer Pharmacology: Case Studies in Dopamine D1 and D2 Receptors

Dopamine receptors mediate various important neurophysiological functions. At a molecular level, G protein coupling is considered the main activation mechanism for most of the receptor-mediated cellular processes. A number of studies using native tissue have supported the idea that receptors can interact to form dimers or higher order oligomers. Particularly in medium spiny neurons of the striatum, dopamine receptor subtypes are reported to form dimers with themselves or other receptors (e.g. adenosine receptor A2A). Although a functional relevance for these dimers has been proposed, current assay systems are not capable of teasing out dimer-specific signaling events from those from other receptor populations. We have developed an assay that allows investigation of receptor-effector coupling specifically with defined dimer pairs. Using this assay, we investigated putative dopamine D1-D2 and A2A-D2 receptor dimer functions and studied the issue of a purported G protein coupling switch in the D1-D2 receptor dimer in which the heteromer was proposed to activate Gq, unlike D1 or D2 receptor when expressed alone. We were unable, however, to find evidence for Gq activation by the D1-D2 heteromer, as the protomers in the heteromer maintained fidelity of signaling to their cognate G proteins. We also developed and optimized a series of novel Gs biosensors to elucidate differences in heterotrimeric G protein conformational changes triggered by dopamine D1 and A2A receptors, two of the prominent pharmacological targets in the striatum. In addition to G protein signaling, intracellular calcium is also involved in many important cellular functions in all cell types. In neurons, intracellular calcium is implicated in learning and memory (synaptic plasticity) as well as neurodegeneration (apoptosis). In medium spiny neurons, dopamine-mediated calcium release from internal stores has been reported to result from activation of phospholipase C (PLC). However, different subtypes of dopamine receptors and intermediary proteins have been proposed to play a role in this dopamine-mediated PLC activation, and the underlying mechanisms are unclear. We found that activation of D1 and D2 receptors expressed individually can mobilize calcium in a PLC-dependent manner. In parallel, we also examined D1 and D2 receptor colocalization in striatal brain slices as well as in cultured medium spiny neurons. Although we found evidence using bacterial artificial chromosome-D1 and D2 reporter mice that D1 and D2 receptors are co-expressed in a small number of brain regions, we failed to observe D1-D2 receptor colocalization, suggesting the possibility that in neurons the receptors are somehow segregated

Development of a Field Test for Evaluating Aerobic Fitness

The purpose of this study was to investigate the reproducibility and utility of a standardised and externally paced field test (15-m Incremental Shuttle Walk and Run Test [15 mISWRT]) to assess aerobic fitness in middle-aged adults. 14 middle-aged participants performed the 15-m ISWRT 3 times within one week (Test 1, Test 2, Test 3). Reproducibility of the 15-m ISWRT was tested by comparing 15-m ISWRT performance (distance completed), HRmax, and V˙O2max for each test. The utility of the 15-m ISWRT for evaluating V˙O2max over a wide range in middle-aged adults was tested by comparing the range of V˙O2max obtained from the portable expired gas analyzer with the V˙O2max reference values and ranges for health promotion published by Japan\u27s Ministry of Health, Labour and Welfare. A multiple comparison of distance completed in the 15-m ISWRT Test 1, Test 2, and Test 3 found no significant difference between Test 2 and Test 3. The ICC was 0.99 for Test 2 vs. Test 3. V˙O2max measured from the 15-m ISWRT in Test 3 had a minimum value of 22.8 ml/kg/min and a maximum value of 38.7 ml/kg/min. In conclusion, the 15-m ISWRT is reliable and useful for evaluating V˙O2max in middle-aged adults

On the Virulence of Mycoplasma gallisepticum Strain 1RF Artificially Induced into the Air Sac of Day-Old Chicks

in vivoでの抗菌剤効果の基礎的検討の為に,初生ヒナを用いた一実験感染系を確立する目的で,以下の実験を試みた。 供試菌Mycoplasma gallisepticum (MG)1RF株の新鮮液体培養10倍段階希釈の0.4ml/ヒナを,ブロイラー初生ヒナ,雄の右後胸部気嚢内に接種し,4週間観察し,接種菌のvirulenceの程度を調べた。実験群は20羽/群とし,非感染対照を含む6群を設定した。接種菌数は4×10^1-5 CCUの5段階とした。各実験群のヒナについては,臨床症状を観察し,増体量,飼料要求率を調べて,毎週各群5羽ずつを殺して剖検し,MG分離,MG血清凝集素価の推移,肉眼ならびに組織病変等について経時的観察を試みた結果,下記の成績が得られた。 1) 全期間,全実験群において呼吸器症状はみられなかった。しかし,接種菌数の多い群で頸部捻転と脚麻痺を主徴とする死亡ヒナがみられ,これらのヒナの脳病変は病理組織学的検査の結果,脳軟化のそれに一致した。それらヒナの一部の脳からはMGが分離された。 2) 接種菌数の多い群では.ヒナの増体量は少なく飼料要求率は悪かった。 3) 接種菌数の少ない群での死亡率は極めて低く,接種菌数の多い群で14日令まで高く,その後28日令までは死亡ヒナはみられなかった。 4) 気嚢および気管の病変出現率は,MG接種菌数の多い群で高く,また病変の程度も強いものが多かった。なお低率ながら各群のヒナに肝の点状出血や,線維素性肝包膜炎,壊死巣などが,MG接種菌数の多少にかかわらず認められた。 5) MG分離率は,気嚢よりも気管において高く,またMG非接種側気嚢よりもMG接種側気嚢において高かった。 6) MG血清凝集素価は,1週令殺時のヒナ群では低く,2週令時以後の殺ヒナ群で上昇がみられた。MG接種群間では接種菌数の多少にかかわらず凝集素価に大きな差は認められなかった。 7) 供試菌株のAID50は10CCU前後とみられた。 結論として,供試1RF株はM. gallisepticumとしては,ヒナに対し比較的強毒な菌株である事が知られた。In order to establish an experimental system using chicks for the fundamental observation on efficacy in vivo of the antibacterial agents that control the avian respiratory mycoplasmosis, the present experiments were conducted to investigate the virulence of Mycoplasma gallisepticum strain 1RF26) induced artificially into the air sac of day-old chicks. The inocula of 0.4 ml/chick of ten-fold serial dilutions of broth culture of the organisms were artificially induced into the right posterior thoracic air sac of day-old male broiler chicks, and the chicks were observed during 4 weeks. The experimental groups were designed as group of 20 chicks each, 6 groups included a non-infected control and 5 infected groups. The inoculum sizes of the organisms to the chicks ranged from 4×10 to 10^5 CCU* in 5 dilutions of the broth culture. The following results were obtained. 1) Clinical symptoms were observed only death with characteristic findings of limberneck and leg weakness in the chicks inoculated with a large number of the organisms. According to the results of the histopathological examinations, the cerebral lesions appearing in these chicks were similar to those of the encephalomalacia. The inoculated organisms were recovered from the brain of some of those chicks. 2) Low weight gains and high feed conversions were observed in the group of chicks inoculated with a large number of the organisms. 3) The mortality rate was very low in the group of chicks inoculated with a small number of the organisms. In the group of chicks inoculated with a large number of the organisms, the mortality of chicks was comparatively high up to 14 days post inoculation, but no mortality was observed during the 15 to 28 days post inoculation. 4) Incidence and severity of macroscopic lesions in the air sac and the trachea of the chicks inoculated with a large number of the organisms were comparatively high and severe. Under low incidence of the macroscopic lesions including petechiae, fibrinous perihepatitis and necrotic foci etc. in the liver of chicks were observed to be not depending on the inoculum size of the organisms. 5) Recovery ratio of the organisms from the chicks was found to be higher in the trachea than that in the air sac, and furthermore was higher on the inoculated side than on the other side of air sac. 6) Serum agglutinins against M. gallisepticum were observed to be at a low titer in the chicks killed 1 week post inoculation, and increased titers were observed in the chicks killed after 2 weeks post inoculation. No remarkable difference of the serum agglutinin titers depending on inoculum size of the organisms was observed in the chicks. 7) The AID50 of the organisms was estimated as ca. 10 CCU. Consequently, it was proved that the strain 1RF examined is comparatively high virulent against chicks, among the strains of M. gallisepticum.本論文の要旨は，第85 回日本獣医学会(1978 年4月)で口頭発表した

Acinar Cell Carcinoma of the Pancreas with Colon Involvement

We report a case of acinar cell carcinoma of the pancreas with colon involvement that was difficult to distinguish from primary colon cancer. A 60-year-old man was admitted with a 1-month history of diarrhea. Contrast-enhanced computed tomography (CT) revealed a large tumor (10.6×11.6 cm) at the splenic flexure of the colon. Colonoscopy showed completely round ulcerative lesions, and biopsy revealed poorly differentiated adenocarcinoma. Left hemicolectomy, resection of the jejunum and pancreas body and tail, and splenectomy were performed based on a diagnosis of descending colon cancer (cT4N0M0, stage IIB), and surgery was considered to be curative. Diagnosis was subsequently confirmed as moderately differentiated acinar cell carcinoma of the pancreas by immunohistochemical staining (pT3N0M0, stage IIA). Multiple liver metastases with portal thrombosis were found 8 weeks postoperatively. Despite combination chemotherapy with oral S-1 and gemcitabine, the patient died of hepatic failure with no effect of chemotherapy 14 weeks postoperatively. Correct diagnosis was difficult to determine preoperatively from the clinical, CT, and colonoscopy findings. Moreover, the disease was extremely aggressive even after curative resection. Physicians should consider pancreatic cancer in the differential diagnosis of similar cases

Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

By analyzing and simulating inactive conformations of the highly-homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands

Expressions of Vascular Endothelial Growth Factor (VEGF)-D and VEGF Receptor-3 in Colorectal Cancer: Relationship to Lymph Node Metastasis

Angiogenic factors play a major role in tumor growth and metastasis. Vascular endothelial growth factor (VEGF)- D is a ligand for VEGF receptor-3 (VEGFR-3/Flt-4), which mainly expressed on the lymphatic endothelium. Recent experimental studies have shown that VEGF-D induces tumor lymphangiogenesis and promote metastatic spread of tumor cells via lymphatic vessels. However, the contribution of VEGFD to lymph node metastasis in human colorectal cancer is less understood. We therefore examined VEGF-D and VEGFR-3 expression in patients with colorectal cancer. Sections of formalin-fixed and paraffin-embedded specimens from 76 colorectal cancers were immunohistochemically stained for VEGF-D and VEGFR-3. Staining for VEGF-D was positive in the cytoplasm of tumor cells in 43 of 76 examined tumors (56.6%). Staining for VEGFR-3 was positive in endothelial cells in 38 (50.0%) tumors. Univariate analysis showed that both VEGF-D and VEGFR-3 expressions correlated significantly with lymph node metastasis, histological type and depth of tumor invasion. However, logistic regression analysis indicated that VEGF-D expression, but not that of VEGFR-3, was an independent predictor for lymph node metastasis. Our data suggest that VEGF-D plays an important role in lymph node metastasis in colorectal cancer

Functional Selectivity of Allosteric Interactions within GPCR oligomers: the Dopamine D1-D3 Receptor Heterotetramer

The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques and cell signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and GI proteins, respectively. Co-activation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1 and to a positive crosstalk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive crosstalk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of YFP fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists