Fig 1.
Dendrogram of structural relatedness for 51 human GPCRs including MT1/MT2.
FASTA protein sequences for 51 randomly sampled GPCRs were structurally predicted using AlphaFold2 and compared using Dali All Against All. MT1/MT2 outlined in red. Color boxes denote GPCR class: Class A/Rhodopsin (blue), Class B/Secretin (red), Class C/Glutamate (yellow), Class F/Frizzled/Taste (green), Class Adhesion (purple), other (no color). Dendrogram formatted using Phylo.io.
Fig 2.
Dendrogram of structural relatedness between 43 fungal GPCRs and MT1/MT2.
FASTA protein sequences for the 43 identified fungal GPCRs and human MT1/MT2 were structurally predicted using AlphaFold2 and compared using Dali All Against All. A sister clade is formed with four fungal GPCRs, GPR-1-4, marked with a green box. The clade containing MT1/MT2 is shown with a red box. Roman numerals indicate which of the 14 fungal GPCR class each GPCR falls into as outlined in Cabrera et al., 2015. Dendrogram formatted using Phylo.io.
Fig 3.
Genetic knockouts of gpr-3 are insensitive to melatonin-induced phase shifts of TTFO negative element FRQ.
Difference in FRQ::LUC between gpr-3- (N380-2, n = 3) and gpr-3+ (N380-1, n = 3) strains of Neurospora crassa two hours after treatment with 5ng/mL melatonin in 1x fructose/glucose/sorbose media (FGS). Control group was treated with sterile water. Two-way ANOVA revealed significant effect of melatonin treatment on N380-1 (p = 0.02) but not in the strain lacking gpr-3 (p = 0.07). FRQ::LUC–luciferase translational reporter for key clock component Frequency, *–p<0.05, ns–not significant.
Fig 4.
Knockouts of gpr-3 lack cAMP signaling response to melatonin.
Difference in relative cAMP concentration between gpr-3+ (N380-1, n = 6), gpr-3- (N380-2, n = 6), and gna-1-(n = 6) strains after 10-minute treatment with 5ng/mL melatonin in sterile water. Untreated group was treated only with water. One-way ANOVA revealed statistically significant decrease of cAMP in response to melatonin treatment in N380-1 when compared to N380-2 (p = 0.036). There was no significant difference between N380-1 or N380-2 with gna-1 (p = 0.204, p = 0.607, respectfully. nd–no difference, *–p<0.05.