1. There are 800 genes in this family in the mammalian genome G-Protein Coupled Receptors

3. Orphan Receptors in the GPCR family Rhodopsin and b-adrenergic receptor found to be homologous “Artisanal Period”: Using known ligands to find their GPCR’s dopamine, adenosine; angiotensin, vasopressin etc “De-orphanisation” Using GPCR to find new transmitters e.g. nociceptin, ghrellin Industrial approach, many ligands and receptors identified olfactory ligands, taste receptors, H +, Ca ++ Still; the majority of GPCR do not have known ligands; so they have been given numbers

4. De-orphanization Protocols Many industrial approaches but most depend on the following Express GPCR of interest in a cell line which has the requisite signaling system, e.g. increase in Ca Use extracts of organs or blood to see if they activate the signal Purify the fraction that does it using this assay

5. Tularik Co, wanted to find the ligands for GPCR 91, and GPCR 99 They are homologous to the Purinergic (ATP) receptors But after much search the activity in the kidney turned out to be Succinate for GPR91 and  -ketoglutarate for GPR99 Nature. 2004 ;429:188-93

6. Krebs Cycle GPR91 GPR99

7. GPR91 GPR99 Highest expression is in the Kidney Mostly Proximal Tubule and JGAMostly Distal Tubule

8. Succ  -KG

9. Normal Nx

11. Isolated Perfused JGA

12. In Isolated Perfused JGA : High Glucose increases renin release

13. In Isolated Perfused JGA : Perfusion of Succinate increases renin release Fluorocitrate

14. Renin release in Isolated Perfused JGA Succinate in the bath is faster than high glucose

15. Succinate Levels in Diabetic Urine and Kidneys

16. Renin Release from Isolated perfused JGA preparation

17. GPR91RECA-1Merged

18. High Glucose and Succinate increase cell Calcium in isolated endothelial cells

19. High Glucose and Succinate increase cell Calcium via GPR91 in isolated glomerular endothelial cells

20. High Glucose or Succinate increase cell Ca In isolated glomeruli

21. High Succinate increase PGE2 production

22. +/+-/- Control STZ Diabetes Diabetes increases renin granule content only in GPR91+ mice