?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.title=In+vitro+and+in+vivo+studies+on+protease-activated+receptor-2&rft.creator=Adams%2C+Mark+N.&rft.subject=protease-activated+receptor-2+(par2)%2C+g-protein+coupled+receptor+(gpcr)%2C+signal+transduction%2C+trafficking%2C+palmitoylation%2C+agonist%2C+antagonist%2C+prostate+cancer%2C+bone+metastasis%2C+scid+mice%2C+intra-tibial+injection&rft.description=Protease-activated+receptor-2+(PAR2)+is+a+G+protein+coupled+receptor+(GPCR)+that+is+activated+by+proteolytic+cleavage+of+its+amino+terminal+domain+by+trypsin-like+serine+proteases.+Cleavage+of+this+receptor+exposes+a+neoepitope%2C+termed+the+tethered+ligand+(TL)%2C+which+binds+intramolecularly+within+the+receptor+to+stimulate+signal+transduction+via+coupled+G+proteins.+PAR2-mediated+signal+transduction+is+also+experimentally+stimulated+by+hexapeptides+(agonist+peptides%3B+APs)+that+are+homologous+to+the+TL+sequence.+Due+to+the+irreversible+nature+of+PAR2+proteolysis%2C+downstream+signal+transduction+is+tightly+regulated.+Following+activation%2C+PAR2+is+rapidly+uncoupled+from+downstream+signalling+by+the+post-translational+modifications+phosphorylation+and+ubiquination+which+facilitate+interactions+with+%C3%A2-+arrestin.+This+scaffolding+protein+couples+PAR2+to+the+internalisation+machinery+initiating+its+desensitisation+and+trafficking+through+the+early+and+late+endosomes+followed+by+receptor+degradation.%0D%0A%0D%0APAR2+is+widely+expressed+in+mammalian+tissues+with+key+roles+for+this+receptor+in+cardiovascular%2C+respiratory%2C+nervous+and+musculoskeletal+systems.+This+receptor+has+also+been+linked+to+pathological+states+with+aberrant+expression+and+signalling+noted+in+several+cancers.+In+prostate+cancer%2C+PAR2+signalling+induces+migration+and+proliferation+of+tumour+derived+cell+lines%2C+while+elevated+receptor+expression+has+been+noted+in+malignant+tissues.+Importantly%2C+a+role+for+this+receptor+has+also+been+suggested+in+prostate+cancer+bone+metastasis+as+coexpression+of+PAR2+and+a+proteolytic+activator+has+been+demonstrated+by+immunohistochemical+analysis.%0D%0A%0D%0ABased+on+these+data%2C+the+primary+focus+of+this+project+has+been+on+two+aspects+of+PAR2+biology.+The+first+is+characterisation+of+cellular+mechanisms+that+regulate+PAR2+signalling+and+trafficking.+The+second+aspect+is+the+role+of+this+receptor+in+prostate+cancer+bone+metastasis.+In+addition%2C+to+permit+these+studies%2C+it+was+first+necessary+to+evaluate+the+specificity+of+the+commercially+available+anti-PAR2+antibodies+SAM11%2C+C17%2C+N19+and+H99.+The+evaluation+of+the+four+commercially+available+antibodies+was+assessed+using+four+techniques%3A+immunoprecipitation%3B+Western+blot+analysis%3B+immunofluorescence%3B+and+flow+cytometry.+These+approaches+demonstrated+that+three+of+the+antibodies+efficiently+detect+ectopically+expressed+PAR2+by+each+of+these+techniques.+A+significant+finding+from+this+study+was+that+N19+was+the+only+antibody+able+to+specifically+detect+N-glycosylated+endogenous+PAR2+by+Western+blot+analysis.+This+analysis+was+performed+on+lysates+from+prostate+cancer+derived+cell+lines+and+tissue+derived+from+wildtype+and+PAR2+knockout+mice.+Importantly%2C+further+evaluation+demonstrated+that+this+antibody+also+efficiently+detects+endogenous+PAR2+at+the+cell+surface+by+flow+cytometry.%0D%0A%0D%0AThe+anti-PAR2+antibody+N19+was+used+to+explore+the+in+vitro+role+of+palmitoylation%2C+the+post-translational+addition+of+palmitate%2C+in+PAR2+signalling%2C+trafficking%2C+cell+surface+expression+and+desensitization.+Significantly%2C+use+of+the+palmitoylation+inhibitor+2-bromopalmitate+indicated+that+palmitate+addition+is+important+in+trafficking+of+PAR2+endogenously+expressed+by+prostate+cancer+cell+lines.+This+was+supported+by+palmitate+labelling+experiments+using+two+approaches+which+showed+that+PAR2+stably+expressed+by+CHO+cells+is+palmitoylated+and+that+palmitoylation+occurs+on+cysteine+361.+Another+key+finding+from+this+study+is+that+palmitoylation+is+required+for+optimal+PAR2+signalling+as+Ca2%2B+flux+assays+indicated+that+in+response+to+trypsin+agonism%2C+palmitoylation+deficient+PAR2+is+~9+fold+less+potent+than+wildtype+receptor+with+a+reduction+of+about+33%25+in+the+maximum+signal+induced+via+the+mutant+receptor.+Confocal+microscopy%2C+flow+cytometry+and+cell+surface+biotinylation+analyses+demonstrated+that+palmitoylation+is+required+for+efficient+cell+surface+expression+of+PAR2.+Importantly%2C+this+study+also+identified+that+palmitoylation+of+this+receptor+within+the+Golgi+apparatus+is+required+for+efficient+agonist-induced+rab11amediated+trafficking+of+PAR2+to+the+cell+surface.+Interestingly%2C+palmitoylation+is+also+required+for+receptor+desensitization%2C+as+agonist-induced+%C3%A2-arrestin+recruitment+and+receptor+degradation+were+markedly+reduced+in+CHO-PAR2-C361A+cells+compared+with+CHO-PAR2+cells.+Collectively%2C+these+data+provide+new+insights+on+the+life+cycle+of+PAR2+and+demonstrate+that+palmitoylation+is+critical+for+efficient+signalling%2C+trafficking%2C+cell+surface+localization+and+degradation+of+this+receptor.+This+project+also+evaluated+PAR2+residues+involved+in+ligand+docking.+Although+the+extracellular+loop+(ECL)2+of+PAR2+is+known+to+be+required+for+agonist-induced+signal+transduction%2C+the+binding+pocket+for+receptor+agonists+remains+to+be+determined.+In+silico+homology+modelling%2C+based+on+a+crystal+structure+for+the+prototypical+GPCR+rhodopsin%2C+and+ligand+docking+were+performed+to+identify+PAR2+transmembrane+(TM)+amino+acids+potentially+involved+in+agonist+binding.+These+methods+identified+12+candidate+residues+that+were+mutated+to+examine+the+binding+site+of+the+PAR2+TL%2C+revealed+by+trypsin+cleavage%2C+as+well+as+of+the+soluble+ligands+2f-LIGRLO-NH2+and+GB110%2C+which+are+both+structurally+based+on+the+AP+SLIGRLNH2.%0D%0A%0D%0ALigand+binding+was+evaluated+from+the+impact+of+the+mutated+residues+on+PAR2-mediated+calcium+mobilisation.+An+important+finding+from+these+experiments+was+that+mutation+of+residues+Y156+and+Y326+significantly+reduced+2f-LIGRLO-NH2+and+GB110+agonist+activity.+L307+was+also+important+for+GB110+activity.%0D%0A%0D%0AIntriguingly%2C+mutation+of+PAR2+residues+did+not+alter+trypsin-induced+signalling+to+the+same+extent+as+for+the+soluble+agonists.+The+reason+for+this+difference+remains+to+be+further+examined+by+in+silico+and+in+vitro+experimentation+and%2C+potentially%2C+crystal+structure+studies.+However%2C+these+findings+identified+the+importance+of+TM+domains+in+PAR2+ligand+docking+and+will+enhance+the+design+of+both+PAR2+agonists+and+potentially+agents+to+inhibit+signalling+(antagonists).%0D%0A%0D%0AThe+potential+importance+of+PAR2+in+prostate+cancer+bone+metastasis+was+examined+using+a+mouse+model.+In+patients%2C+prostate+cancer+bone+metastases+cause+bone+growth+by+disrupting+bone+homeostasis.+In+an+attempt+to+mimic+prostate+cancer+growth+in+bone%2C+PAR2+responsive+22Rv1+prostate+cancer+cells%2C+which+form+mixed+osteoblastic+and+osteolytic+lesions%2C+were+injected+into+the+proximal+aspect+of+mouse+tibiae.+A+role+for+PAR2+was+assessed+by+treating+these+mice+with+the+recently+developed+PAR2+antagonist+GB88.+As+controls%2C+animals+bearing+intra-tibial+tumours+were+also+treated+with+vehicle+(olive+oil)+or+the+prostate+cancer+chemotherapeutic+docetaxel.+The+effect+of+these+treatments+on+bone+was+examined+radiographically+and+by+micro-CT.%0D%0A%0D%0AConsistent+with+previous+studies%2C+22Rv1+tumours+caused+osteoblastic+periosteal+spicule+formation+and+concurrent+osteolytic+bone+loss.+Significantly%2C+blockade+of+PAR2+signalling+reduced+the+osteoblastic+and+osteolytic+phenotype+of+22Rv1+tumours+in+bone.+No+bone+defects+were+detected+in+mice+treated+with+docetaxel.%0D%0A%0D%0AThese+qualitative+data+will+be+followed+in+the+future+by+quantitative+micro-CT+analysis+as+well+as+histology+and+histomorphometry+analysis+of+already+collected+tissues.+Nonetheless%2C+these+preliminary+experiments+highlight+a+potential+role+for+PAR2+in+prostate+cancer+growth+in+bone.%0D%0A%0D%0AIn+summary%2C+in+vitro+studies+have+defined+mechanisms+regulating+PAR2+activation%2C+downstream+signalling+and+trafficking+and+in+vivo+studies+point+to+a+potential+role+for+this+receptor+in+prostate+cancer+bone+metastasis.+The+outcomes+of+this+project+are+that+a+greater+understanding+of+the+biology+of+PAR2+may+lead+to+the+development+of+strategies+to+modulate+the+function+of+this+receptor+in+disease.&rft.publisher=Queensland+University+of+Technology&rft.date=2012&rft.type=Thesis&rft.format=application%2Fpdf&rft.relation=https%3A%2F%2Feprints.qut.edu.au%2F54338%2F1%2FMark__Adams_Thesis.pdf&rft.rights=free_to_read&rft.relation=Adams%2C+Mark+N.+(2012)+In+vitro+and+in+vivo+studies+on+protease-activated+receptor-2.+PhD+thesis%2C+Queensland+University+of+Technology.&rft.id_number=https%3A%2F%2Feprints.qut.edu.au%2F54338%2F&rft.identifier=Faculty+of+Health