Molecular And Mechanistic Characterization Of GPCR-mediated CREB Signaling Pathway And Non-visual Arrestin From Silkworm, Bombyx Mori

The cAMP response element-binding protein, CREB, is a GPCR signal activated transcription factor implicated in the control of many biological processes, such as spermatogenesis, memory and circadian rhythms. CREB is phosphorylated at serine residue133by cAMP-dependent protein kinase A, resulting in initiation of transcription of CREB target genes by recruiting the coactivators CBP or p300that associate with the RNA polymerase Ⅱ complexes. CRE-driven luciferase expression has been widely used to detect the cAMP elevation, but the vector used in mammalian cell is not suitable for insect cells. In the current study, a CRE-driven luciferase assay system was constructed for GPCR characterization in insect cells, our data indicated that Bombyx Gs-coupled adipokinetic hormone receptor and corazonin receptor could effectively initiate CRE-driven luciferase transcription, but forskolin, a reagent to be widely used for activating adenylyl cyclase in mammalian system, failed to induce CRE-driven luciferase expression in insect cells transfected with a CRE-driven reporter construct. Further investigation revealed that PKC specific inhibitor exhibited stimulatory effect on the CRE-driven reporter transcription, whereas blockade of Ca2+signal and inhibition of Ca2+-dependent calcineurin resulted in a significant decrease on the expression of luciferase in insect cells. These results suggest that PKC is likely as a negative regulator to modulate CREB activation, in contrast, other than PK.A, Ca2+signal and Ca2+-dependent calcineurin also essentially contribute to the positive regulation of CREB activity. In addition, the extracellular signal-regulated kinase (ERK) has no significant effect on CRE-driven luciferase expression.As their names imply, Arrestins "arrest" the agonist to activate GPCR. This belrivor has been widely applied to GPCR signaling detection and ligand screening. Artestins are divided into two groups:visual arrestins turn off the Rhodopsin signaling specilicly, while as important scaffold protein, the non-visual arrestins (β-aneslin1/2) ate distributed more widely and related to receptor internalization, ERK phosphorylation. Rho/ROCK signaling, interleukins/tumor necrosis factor production, activation of NF-κB and so on, besides GPCR desensitization. Referring to non-visual arrest in of Drosophila melanogaster, DmKurtz, we obtained the cDNA sequence containing a coding sequence of kurtz of Bomhyx mori by ESTs (expression sequence tags) splicing and RACE (rapid amplification of cDNA ends) and named after DmKurlz. Sequence alignment showed that BmKurtz contained the non-visual arrestin specific domains, and the result of real-time PCR demonstrated that BmKurtz was distributed widely in5th instar day-3larvae. Furthermore, just like DmKurtz, the BmKurtz could also be recruited by activated AKHR, and the ERK phosphorylation was delayed significantly by over-expression of BmKurtz.Here we modified the CRE-Luc vector and to make it suitable for GPCR signaling in lepidopteran cell lines. Furthermore, our study shed new light on the underlying molecular mechanism by which CREB activation is regulated in insect. The identification of BmKurtz is an important supplement of GPCR signaling detection system.