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

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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. Introduction: discovery of GPR61 in adrenal. Aldosterone producing adenomas (APA) cause ~4% of hypertension. - PowerPoint PPT Presentation

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  • 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

  • Introduction: discovery of GPR61 in adrenal

    Fold ChangeGENEBayes.pGPR617.65E-067.6ITGB1BP32.71E-056.3MYB1.14E-115.9BAPX13.67E-055.8KCNK48.15E-065.5CCL220.0001884.8ENO31.16E-054.7ATP2A31.27E-064.6MAT1A7.78E-054.5PVALB9.55E-054.5HTR44.62E-054.4CACNB23.60E-054.4

  • 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 genomesEndogenous 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).AdenylateCyclaseATPcAMPGAldosterone

  • 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:To replicate microarray findingsTo detect expression of GPR61 at protein levelTo determine anatomical localisation of GPR61 in-vitroIntervention Studies: To determine whether GPR61 blockade may influence aldosterone secretion

  • MethodsGPR61 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 qPCR0.003 (1.27)0.42 (1.08)

  • Results: Quantification of GPR61 Protein by Western BlotsWestern Blot Densitometry (arbitrary units)N1 T1 N2 T2 N3 T3 N4 T4NormalTumourN=4

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

  • Results: Functional Studies using 4h-aldosterone secretion from primary adrenal cellsN=5P= 0.004

  • Summary and ConclusionGPR61 is expressed in adrenal cortex, ZG and ZFPreliminary studies with 5-NOT indicate possible constitutive role in regulating aldosterone secretion.

    Future work: de-orphanisation of GPR61 as a drug target, using -arrestin-pathway recruitment detection systemFunctional studies: Does GPR61 contribute to aldosterone secretion in vivo?

    Good Afternoon, my name is Lalarukh Haris and today I will present to you my MPhil project which is supervised by Dr. Anthony Davenport and Prof. Morris Brown. The topic of my project is GPR61, an orphan G-protein coupled receptor which is a potential candidate for the regulation of aldosterone secretion.*15-25% of patients with hypertension have primary hyperaldosteronism or conns disease, without proof of laterlising adenoma. Aldosteronomas are curable aldosterone-secreting benign tumours of the adrenal cortex From 20,000 genes that were screened in a previous microarray study carried out by our group, eight aldosteronmas were compared with paired normal adrenals from the same patients. Out of this about 7-8 genes were the top hits. These included potassium chanels, serotonin receptor, cytochrome p450, solute carrier and GPR61. GPR61 is the basis of my project- it is novel but most importantly a drugable orphan target that codes for a G-protein receptor (a seven transmembrane domain receptor). It gave a 8 fold change increase in tumour vs. the normal adrenal. *So lets go back to the basis- What do we actually know about this protein so far? And the answer to that is not very much. We know that it is present in all genomes ( point picture mouse!-y-axis avg exp. X-axis tissues distribution by qpcr high exp of mrna in brain but also some expression in adrenal gland we can see that it is abundantly present in the brain and in the adrenal gland). We know it is one of the 93 remaining class A orphan receptors, which means its endogenous ligand is not yet known. Secondary messanger maybe active in cAMPIt is constituatively active and predicted that its surrogate ligand amine receptor is 5NOT which is a low infinity inverse agonist. *So keeping this in mind it became very interesting for me to hypothesis that the up-regulation of GPR61 in aldosterone producing adenomas leads to over production of the camp which plays a role in causring Conns disease and therefore tumour formation this is of course very clinically relevant as it represents a novel target for drug intervention by agonist and antagonists.

    The aims of the project were divided in terms of pharmacological studies and functional studies.Could I replicate the finding of microarray study that was carried out previous by our group?Is gpr61 then expressed at a protein level?To localise GPR61 in terms of in vivo zonation of the adrenal gland.And functionally, is GPR61 constitutively active in native adrenal cells or following over expression.

    *For my methodolgy : Mphil learnt different techniquesI used taqman qpcr to replicate the expression level of GPR61 in 22pairs of tumour and normals I then carried out western blots to quantify the protein expression level in both tumour and normal samplesI localised GPR61 in both tumour and normal whereby assesing the the localisation in terms of adrenal zonation by immunohistochemistryAldosterone assay was carried out on primary adrenal cell cultures which were treated with 5NOT which is sought to be the low affinity inverse agonist. *Quantitative western blots were carried out to assess the expression of gpr61 on a protein level. Raw data, house keeping gene protein at right size. Here we interestingly/surprisingly find that GPR61 is being expressed less in tumour vs. The normal. This suggests that perhaps the protein is being internalised in aldosterone secreting tumours. This is interesting and suggests that GPR61 must play a role aldosterone secreting tumours. At protein level being expressed intensity being measuredConsistent trend throughout surprising Opp direction to mRNA level eg. Modulation silencing disconnct between rna and protein over expression of endogenous ligand speculation why mrna diff to protein level.

    -further confocal to see internalisation also barrestin assay to see protein protein interactions

    *Immunohistochemistry was carried out using rabbit polyclonal Abcam C-terminal antibody and stained with peroxidase which gives a brown stain as can be seen in the image. So as many of you may know the adrenal consists of 3 main layers (go through layers) , to charecterise our protein it is important to understand where it is expressed. From our icc we can see the the abundancy of gpr61 is expressed on the periperal layer namely Zona glomerulosa. We can also say that it is slightly difference in its expression in tumour vs. Normal. We can see diffuse protein expression the tumour. We also observed that the ihc on adrenals with high grp61 expression on qpcr was consistent with predominantly zg distribution which is extremely interestingHigh immunoreactivity less in other layers right hand side control Presence of stainCan localise*So the finding that a constitutively active Gs-coupled receptor is switched on, at least at RNA level, in most Conns adenomas offers a putative explanation for either or both of the enhanced aldosterone secretion, and development of adrenal nodules. Pathway analysis of the microarray results has not yielded any strong clues to the identity of an endogenous ligand (if there is one), or to whether up- or down-regulation of other genes is responsible for GPR61 being switched on in adenomas. It is intriguing that it has been considered a biogenic amine receptor-like receptor The closest receptor, sequence-wise, is the 5HT6, and the constitutive activity of the expressed GPR61 allowed recognition of a 5-(nonyloxy)-tryptamine as a low-affinity inverse agonist.1,8 It remains unlikely, of course, that GPR61 is the sole cause of aldosteronoma formation either overall or in an individual patient. However it is worthy of investigation because of the clues this may provide to regulation of aldosterone secretion, of adenoma formation, and the role of this largely unstudied orphan GPCR. In addition, there is clinical interest in determining whether variation among tumours in GPR61 expression correlates with functionally important differences, such as differentiation into an aldosterone-secreting tumour versus growth as an incidentaloma, or response of a patients hypertension to adrenalectomy. Here we can see that the increased concentration of 5NOT agonist is causing a noticable decrease in aldosterone secretion which highlights our prediction that is may have a 5not type ligand.

    Too low- nasty side effects ht2b and ht4 -not consistent hard to plate cells. no good way to normalise as fibroblasts. *GPR61 is a novel therapeutic target which is in the right place and right time to potentially play a role in cAMP pat