Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: GPR61 gene locus is associated with (A) BMI and (B) weight at genome-wide level of significance. The plots show all the GPCR genes found in the top 1000 genes for each of the 2 associations as analyzed with the CMDKP. Please see the section for more details.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques: Genome Wide

Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: Protocol for exon sequencing and SNP variant calling. The same procedure was used for GPR61 and MC 4 R.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques: Sequencing, Variant Assay

Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: UK10K obesity screen reveals presence of GPR61 mutations. (A) Mutational landscape of GPR61 in normal population and then the same mutations in severe obesity patients. The data are presented as frequencies. The allele counts can be found in Supplemental Fig. 6 . (B) GPR61 mutations mapped onto a 2-dimensional model of GPR61.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques:

Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: Severe obesity–associated mutations of GPR61 have no statistically significant impact on cell surface trafficking of the overexpressed receptor. NanoBiT-based measurement of total cell and cell surface expression of 34 mutants of GPR61. Data were analyzed for differences between the mutants with the WT by one-way ANOVA with Dunnett’s post hoc analysis. Data are presented as mean of n = 3 independent experiments ± SD. Significance levels are given as: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques: Expressing

Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: Severe obesity–associated mutations of GPR61 have an impact on cAMP production and G s translocation. (A) EPAC-derived FRET-based biosensor reveals that 3 mutations lead to the statistically significant reduction in cAMP production elicited by overexpression of GPR61 in the absence of an agonist (increase in the FRET ratio reflects decrease in cAMP levels). Data are presented as the mean of n = 4 independent experiments ± SD. (B) The presence of overexpressed GPR61 R236C 5.66 leads to a statistically significant difference in the bystander BRET ratio between G α s-67- R luc2 and rGFP-CAAX in comparison with the WT. The reduction in ebBRET signal vs pcDNA3.1 is indicative of G s translocation away (activation) from the cell membrane. The receptor plasmids were overexpressed at 3 different levels to account for any differences in their expression and to confirm that higher receptor expression leads to an increase in its constitutive activity. Data are presented as mean of n = 3 independent experiments ± SD. (C) The presence of overexpressed GPR61 R236C 5.66 led to statistically significant differences in the bystander BRET ratio between G α s-67- R luc2 and rGFP-giantin in comparison with the WT. Data were analyzed for differences between the mutants with the WT by one-way ANOVA with Dunnett’s post hoc analysis. Data are presented as mean of n = 3 independent experiments ± SD. Significance levels are given as: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques: Translocation Assay, Derivative Assay, Over Expression, Comparison, Activation Assay, Membrane, Expressing, Activity Assay