Elucidation Of Mechanism(s) Involved In The G Protein-coupled Receptor-mediated Activation Of Protein Kinase C And Its Downstream Signaling

In the 1980s,protein kinase C?PKC?attracted widespread attention for its role as a DAG-sensitive enzyme and as a"receptor"for potent tumor-promoting factor Phorbol esters.The discovery of PKC resolved a 25-year scientific mystery about how agonists that promote lipid hydrolysis alter cellular physiology.PKC belongs to the Ser/Thr protein kinase family,and its activity is controlled by the reversible release of self-inhibiting pseudosubstrate.Under the stimulation of agonists,G protein coupled receptors?GPCRs?can transmit extracellular signals to intracellular,triggering a series of physiological activities.One of the most important aspect is that it can activate ERK1/2 through PKCs,thereby regulating cell differentiation,mitosis,proliferation and even some abnormal physiological processes,such as the tumorigenesis.Therefore,it is particularly important to explore the signal transduction mechanism of PKC-dependent ERK activation mediated by GPCRs.Firstly,the study in this paper used bioinformatics methods to mine the relevant genes of Kisspeptin and its receptor from the genomic data of A.japonicus,and conducted functional identification and research analysis of the system at the cellular level.The gene encoding the AjKiss precursor translates into a 180-amino acid precursor peptide that produces two mature C-terminal amylated peptides,the one is AjKiss1a?32 amino acids?and the other is AjKiss1b?18 amino acids?.The cDNA sequences of two AjKiss receptors were found in the gene database of A.japonicus and cloned from ovarian tissue by RT-PCR.Functional experimental analysis showed that both AjKissR1 and AjKissR2 expressed and located on the cell membrane,and could rapidly mobilize intracellular Ca²⁺under the action of AjKiss1a and AjKiss1b,but showed certain ligand selectivity?AjKiss1a tended to act on AjKissR2,and AjKiss1b tended to act on AjKissR1?.In addition,under the action of AjKiss1a/b,both AjKissR1and AjKissR2 could mediate the receptor internalization and shared the Ca²⁺signaling pathway depends on G?q-PLC.Further studies found that,the C-terminal core deceptide of AjKiss1b,namely AjKiss1b-10,could activate the G?q-PLC-dependent Ca²⁺signaling pathway,mediate receptor internalization and activate ERK1/2 signaling pathway of AjKissR1/2.Under the action of AjKiss1b-10,both AjKissR1 and AjKissR2could activate ERK1/2 via G?q-PLC-Ca²⁺/PKC pathway.However,AjKissR1 could also couple G?i/o and activate ERK1/2 via G??and PKC?dependent pathway.Secondly,this study reported that BomCAPA-PVK-R1?341,a natural splicing variant produced by variable splicing,was a specific receptor for CAPA-PK for the first time.The BomCAPA-PVK-R1 gene consists of seven exons,and the loss of the seventh exon leads to the production of the C-terminal truncated splicing variant,?341.The C-terminal truncation affected the membrane localization of?341,but also affected the GRKs-dependent internalization.Our previous studies on R1 showed that?341 could be used as a negative regulatory protein to affect the membrane localization and function of wild-type receptor R1,but it could not be activated by CAPA-PVKs.However,in this study,?341 was found to be specifically activated by another neuropeptide,CAPA-PK,and mediated downstream signaling pathways.Functional analysis of the experimental results showed that different from G?q coupling wild-type receptor R1,?341 coupled to G?i/o and mediated the down-regulation of intracellular cAMP by the inhibition of adenylate cyclase and the phosphorylation of ERK1/2 via G??-PI3K-PKC?signaling pathways.Taken together,our findings uncover the existence and novel function of the Kisspeptin system in a non-chordate species,and provide not only evidence to support the ancient origin of the hypothalamic neurosecretory system but also an important reference for the research of invertebrate neuropeptides.And the study of a natural splicing variant?341 provides strong evidence for distinguishing the roles of CAPA-PK and CAPA-PVK neuropeptide signaling systems in silkworms and highlights the potential of this knowledge for molecular,pharmacological and physiological studies on GPCR splice variants in the future.