is gpr61, an orphan g protein-coupled receptor, a candidate regulator of aldosterone secretion?

Is GPR61, an orphan G Protein-Coupled Receptor, a candidate regulator of aldosterone secretion?

Lalarukh HarisElena Azizan, Junhua Zhou, Rhoda Kuc, Anthony Davenport &

Morris Brown

• 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

CT PET Overlay

Introduction: discovery of GPR61 in adrenal

Fold ChangeGENE Bayes.p

GPR61 7.65E-06 7.6ITGB1BP3 2.71E-05 6.3MYB 1.14E-11 5.9BAPX1 3.67E-05 5.8KCNK4 8.15E-06 5.5CCL22 0.000188 4.8ENO3 1.16E-05 4.7ATP2A3 1.27E-06 4.6MAT1A 7.78E-05 4.5PVALB 9.55E-05 4.5HTR4 4.62E-05 4.4CACNB2 3.60E-05 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’)

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).

C

N

G G

C

NN

GG G G

AdenylateCyclase ATP

cAMP

Gα Aldosterone

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 findings2.To detect expression of GPR61 at protein level3.To determine anatomical localisation of GPR61 in-vitroIntervention Studies: To determine whether GPR61 blockade may influence aldosterone secretion

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

Results: replication of microarray by qPCR

Normal APA-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5R

elat

ive

abun

danc

e

0.003 (1.27)

0.42 (1.08)

Results: Quantification of GPR61 Protein by Western Blots

GAPDH (36 kDa)

GPR61 (49 kDa)

Wes

tern

Blo

t Den

sito

met

ry

(arb

itrar

y un

its)

N1 T1 N2 T2 N3 T3 N4 T4

NormalTumour

N=4

Results: Localisation of GPR61 by Immunohistochemistry (IHC)

N=9

Results: Functional Studies using 4h-aldosterone secretion from primary adrenal cells

0 10-9 10-7 10-5

5-N-O-T (molar)

-400

-200

0

200

400

600

800

1000

1200A

ldos

tero

ne (p

g/m

L)

N=5P= 0.004

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?