2008 09 09 qais succinate

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					             G-Protein Coupled Receptors




There are 800 genes in this family in the mammalian genome
                 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
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
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 a-ketoglutarate for GPR99




                          Nature. 2004 ;429:188-93
        Krebs Cycle




GPR91                 GPR99
                 Highest expression is in the Kidney


      GPR91                                 GPR99




Mostly Proximal Tubule and JGA                 Mostly Distal Tubule
Succ




a-KG
Normal


Nx
Isolated Perfused JGA
In Isolated Perfused JGA : High Glucose increases renin release
In Isolated Perfused JGA :
Perfusion of Succinate increases renin release




                                                 Fluorocitrate
Renin release in Isolated Perfused JGA
Succinate in the bath is faster than high glucose
Succinate Levels in Diabetic Urine and Kidneys
Renin Release from Isolated perfused JGA preparation
GPR91   RECA-1   Merged
High Glucose and Succinate increase cell Calcium in isolated endothelial cells
High Glucose and Succinate increase cell Calcium via GPR91 in
isolated glomerular endothelial cells
High Glucose or Succinate increase cell Ca
In isolated glomeruli
High Succinate increase PGE2 production
           Diabetes increases renin granule content only in GPR91+ mice




Control




STZ
Diabetes




                +/+                -/-

				
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