Figure 2 : Opsin-Gs-cAMP assay.

From: Cubozoan genome illuminates functional diversification of opsins and photoreceptor evolution

Figure 2

Light activation of opsin-Gs-cAMP pathway by selected opsins. GloSensor™-20F cAMP HEK293 cells (Promega) were transfected with expression vectors encoding genes for different opsins, treated and stimulated with light, as described in Materials and Methods. Arrows represent simple light pulses, multiple arrowheads represent repeated stimulation. Each graph represents a mean of triplicates for every sample. A) Previously reported Gs-cAMP pathway stimulating opsin from C. rastonii (Caryb)27 showed ability to increase the cAMP level in our setup (visualized with cAMP-dependent luciferase activity). The exact homolog of Caryb from T. cystophora Tcop13 showed a highly similar response in our assay. Opsin RH1 from medaka, expected to signal via Gt leading to cGMP decrease, showed no change in luciferase activity. B)–E) Examples of different Tcop light responses. Tcop5 showed faster and weaker activation of the Gs-cAMP pathway than Tcop13. Tcop18 did not activate the Gs-cAMP pathway. F) Analysis of tripeptide activity in Tcop13 was performed. Tcop13 tripeptide HKQ was replaced with tripeptides NKQ, SKS and NRS (originally found in opsins Tcop1 or bovine rhodopsin, Tcop14 and Tcop18 – none of which activated the Gs cascade). Tripeptide mutation did not disrupt Gs activation by Tcop13, but influenced length or sensitivity of Tcop13 response to light stimulation. NT – non-transfected cells used as negative control; Caryb – signal for cells transfected with a vector expressing opsin from C. rastonii, used as positive control; RH1 – signal for cells transfected with a vector expressing opsin RH1 from medaka fish Oryzias latipes, used as negative control; Tcop5, Tcop13, Tcop18 – signal for cells transfected with vectors expressing opsins from T. cystophora - Tcop5, Tcop13 or Tcop18, respectively; NKQ, SKS, NRS – Tcop13 original tripeptide HKQ replaced with tripeptides NKQ, SKS or NRS.