Functional Characterization Of Biogenic Amine Receptors During Different Developmental Stages Of The Pacific Oyster,Crassostrea Gigas

G protein-coupled receptors?GPCRs?compose a large family of membrane receptors and are considered as the largest family of cell-surface receptors.As the mediated moleculars,GPCRs transduct extracellular signals into cells via binding of G protein.GPCRs are involved in various important physiological activities,including sense of sight,taste and smell,regulation of emotion,control of behavior,regulation of nervous system,endocrine system and immune system.Therefore,GPCRs have been widely used in pharmaceutical industry as the target molecular for drugs.Most of the GPCRs belong to the Rhodopsin family?Class A?in living organisms.In the Rhodopsin family,biogenic amine receptors,such as octopamine?OA?receptor,tyramine?TA?receptor,dopamine?DA?receptor,epinephrine receptor and serotonin receptor,are involved in various important vital movements in both vertebrates and invertebrates.Thus,biogenic amine receptors have gained considerable attention from many scientists.Biogenic amine receptors can bind with biogenic amines.They exist in plants and animal,especially in many kinds of food.The molecular weight of biogenic amine is usually not very high.As the putative neurotransmitter,biogenic amines are involved in various important physiological activities,including growth,maturation,and reproduction.OA and its precursor TA are common biogenic amines in invertebrates.They are involved in numerous developmental process of invertebrates such as reproduction,maturation and metamorphosis,and play very important role in these vital process.Although OA was first discovered in octopus?mollusks?,followed studies on OA,TA and related receptors have primarily been conducted in Ecdysozoa,especially in insects.While in mollusks such as bivalve molluscs,only limited reports are available and their physiological roles remain unclear.In this dissertation,we mainly conducted the physiological functional characterization of OA/TA during different developmental stages of the Pacific oyster,Crassostrea gigas.?.Classification and expression profile analysis of GPCRs in C.gigas.Based on the complete genome sequences of C.gigas previously obtained from our research group,about 553 putative GPCRs were screened,identified and annotated from C.gigas.Subsequently,via hydrophobic transmembrane area analysis and family domain screening,412 GPCRs were isolated and identified.By means of BLAST homologous alignment and phylogenetic analysis,the classification of these sequences was finally determined.Among these sequences,308 GPCRs were classified as Class A;44 GPCRs were classified as Class B;14 GPCRs were classified as Class C and 46 GPCRs belong to the unknown family of GPCR protein.In addition,by statistical analysis of the Class A GPCRs in C.gigas,at least 36 GPCRs belong to the subfamily of biogenic amine receptors.Subsequently,via expression profile analysis from different developmental stages of C.gigas,the expression patterns of these GPCRs were classified into 4 types:?1?no expression or very low level of expression for GPCRs was detected during all the developmental stages of C.gigas;?2?GPCRs were mainly expressed in the early developmental stages of C.gigas;?3?GPCRs were mainly expressed in the mid-developmental stages of C.gigas;?4?GPCRs were specificly expressed in some developmental stages of C.gigas.Notably,2 novel biogenic amine receptors?CGI₁0017568 and CGI₁0027100?were also identified from the fourth type,which were considered to involve in the metamorphosis process of C.gigas.?.Functional Characterization of novel OA/TA receptor CgGPR1 during different developmental stages of C.gigas.Via PCR and RACE,a full-length cDNA of 2,826 bp and encoding a putative 524 amino acid OA/TA receptor?CgGPR1,CGI₁0017568?was isolated from C.gigas.According to the BLAST results,CgGPR1 was most closely related to the Lymnaea stagnalis OA receptor OAR2 in sequence.Phylogenetic analysis showed that CgGPR1 belongs to a poorly studied subfamily of invertebrate OA/TA receptors.Subsequently,the spatio-temporal expression of CgGPR1 in different developmental stages and adult tissues of C.gigas was examined by quantitative real-time PCR,Western Blot and whole-mount immunoreactive fluorescence analysis.The results showed that CgGPR1 was expressed during all developmental stages of C.gigas with higher levels at the gastrula stage,trochophore stage and D-shaped larva stage,indicating its potential role in embryogenesis and tissue differentiation.Immunoreactive fluorescence of CgGPR1 was mainly observed in the velum,foot,gill and mantle of C.gigas larvae,indicating its potential role in the swimming,filter-feeding and responding to external environment of C.gigas larvae.Immunoreactive fluorescence of CgGPR1 was also observed in the foot of pediveliger larva stage,indicating its potential role during the metamorphosis of C.gigas.CgGPR1 transcripts were detected in all the tested organs of adult C.gigas,with highest level in the mantle and lowest level in the gonad,which suggested that CgGPR1 may also play roles in the the organs of adult C.gigas.Moreover,by establishing HEK293 cell lines that could stably express CgGPR1,the influence of CgGPR1 to the level of cAMP and Ca²⁺ in HEK293 cells expressing CgGPR1 was conducted with biogenic amine agonists.Pharmacological analysis indicated that cAMP and Ca²⁺ concentrations remained unchanged in HEK293 cells expressing CgGPR1 upon addition of OA,TA or related amines,implying that CgGPR1 uses other unknown molecules as second messenger rather than cAMP and Ca²⁺.Our study lays the foundation for further research of the physiological function of CgGPR1 and its homologous proteins in invertebrates,and provides a potential target molecular for the prevention of marine fouling.?.Spatio-temporal expression pattern analysis of novel TA receptor CgTAR1 during different developmental stages of C.gigas.A full-length cDNA of 1,601 bp and encoding a putative 407 amino acid TA receptor?CgTAR1,CGI₁0027100?was also isolated from C.gigas.According to the BLAST results,CgTAR1 was most closely related to the Locusta migratoria TA receptor TAR1 in sequence.Phylogenetic analysis showed that CgTAR1 belongs to the ?2-adrenergic like tyramine subfamily of invertebrate OA/TA receptors.Afterwards,the spatio-temporal expression of CgTAR1 in different developmental stages and adult tissues of C.gigas was examined by quantitative real-time PCR,Western Blot and whole-mount immunoreactive fluorescence analysis.The results showed that CgTAR1 transcripts were highly expressed in the morula stage,blastula stage and pediveliger larva stage and poorly expressed in the D-shaped larva stage and umbo larva stage.While at the protein level,CgTAR1 was highly expressed from the egg stage to D-shaped larva stage,indicating its potential role in embryogenesis,tissue differentiation and organ formation process of C.gigas.Immunoreactive fluorescence of CgTAR1 was firstly observed in the membrane of many cells in the gastrula stage of C.gigas,and then in the gut of the trochophore stage.Subsequently,immunoreactive fluorescence of CgTAR1 was mainly observed in the velum,umbo and gill,indicating its potential role in the responding to external environment of C.gigas larvae.CgTAR1 transcripts were detected in all the tested organs of adult C.gigas,with highest level in the gonad and lowest in the digestive gland,which indicated that CgTAR1 may also play roles in the the organs of adult C.gigas.Our study sheds light on the physiological function of CgTAR1 in oysters.