1. Is GPR61, an orphan G Protein-Coupled Receptor, a candidate regulator of aldosterone secretion? Lalarukh Haris Elena Azizan, Junhua Zhou, Rhoda Kuc, Anthony Davenport & Morris Brown

2. Aldosterone producing adenomas (APA) cause ~4% of hypertension. Although a minority component of low-renin hypertension, their study may explain inappropriate aldosterone secretion in the majority. In our microarray study of 19594 genes, in eight APA and paired normal adrenal, GPR61 was one of the significant hits in the cluster of genes which included the MC2R (ACTH receptor) Introduction: discovery of GPR61 in adrenal

3. Fold Change GENEBayes.p GPR617.65E-067.6 ITGB1BP32.71E-056.3 MYB1.14E-115.9 BAPX13.67E-055.8 KCNK48.15E-065.5 CCL220.0001884.8 ENO31.16E-054.7 ATP2A31.27E-064.6 MAT1A7.78E-054.5 PVALB9.55E-054.5 HTR44.62E-054.4 CACNB23.60E-054.4

4. What is GPR61? GPR61 is an orphan G-protein receptor which constitutively activates adenylate cyclase – the principal intracellular activator of aldosterone synthase. Mutational activation of cAMP well recognised in benign tumours, including the multiple adrenal nodules of ACTH-independent macronodular hyperplasia (‘AIMAH’)

5. Previous information about GPR61 Present in human, rat, mouse genomes Endogenous ligand predicted to be ‘brain amine like’ Low-affinity binding to 5-nonyl-oxy-tryptamine (5-N-O-T) (Coughlin S.R. et al., Cell, 2008). Adenylate Cyclase ATP cAMP GαGα Aldosterone

6. Hypothesis : Up-regulation of GPR61 in APAs leads to over production of cAMP which plays a role in causing hyperaldosteronism and tumour formation. Aims : Descriptive Studies: 1.To replicate microarray findings 2.To detect expression of GPR61 at protein level 3.To determine anatomical localisation of GPR61 in-vitro Intervention Studies: To determine whether GPR61 blockade may influence aldosterone secretion

7. Methods GPR61 expression was quantified by qPCR in 22 pairs of snap-frozen APA and adjacent normal adrenal. Translation to protein was assessed and quantified by Western blots in paired samples that expressed high levels of GPR61 mRNA Localisation of GPR61 in APA and adjacent normal adrenal was assessed by immunohistochemistry (IHC) Preliminary evidence of GPR61 regulation of aldosterone secretion was sought using the low-affinity 5HT-like ligand 5- (nonyloxy)-tryptamine (5-N-O-T) in primary adrenal cell cultures by carrying out aldosterone assays

8. Results: replication of microarray by qPCR 0.003 (1.27) 0.42 (1.08)

9. Results : Quantification of GPR61 Protein by Western Blots GAPDH (36 kDa) GPR61 (49 kDa) Western Blot Densitometry (arbitrary units) N1 T1 N2 T2 N3 T3 N4 T4 Normal Tumour N=4

10. Results: Localisation of GPR61 by Immunohistochemistry (IHC) N=9

11. Results: Functional Studies using 4h-aldosterone secretion from primary adrenal cells N=5 P= 0.004

12. Summary and Conclusion GPR61 is expressed in adrenal cortex, ZG and ZF Preliminary studies with 5-NOT indicate possible constitutive role in regulating aldosterone secretion. Future work: de-orphanisation of GPR61 as a drug target, using β-arrestin-pathway recruitment detection system Functional studies: Does GPR61 contribute to aldosterone secretion in vivo?