maglaugsch.pdf [1.95 mb] |Untersuchungen zur Dimerisierung von Somatostatin-Rezeptoren mit anderen G-Protein-gekoppelten Rezeptoren|
|Heterodimerization G-Protein-Coupled Receptors somatostatin receptor internalization oligomerization dimerization homodimerization GPCR desensitization immunoprecipitation|
The heterodimerisation of eleven combinations of somatostatin receptor 2A (sst2A) with different G-Protein-Coupled Receptors (GPCRs) was systematically investigated. To achieve this, HEK293 cells (human embryonic kidney cells) expressing sst2A were transfected with the following receptors: sst1-sst5 (somatostatin family), and MOR1 (Opioid family) as class I of GPCR; vesoaciv-intestinal-peptid receptors (VPAC1 and VPAC2) and neurotensin receptors (NT1 and NT2) as class II; and metabotropic glutamate receptors (mGluR1 and mGluR5) as class III. The homodimerisation of sst2A and heterodimerisation of sst2A with MOR1 were proven in living HEK293 cells by means of BRET (Bioluminecence resonance energy transfer) as well as in isolated receptors through co-immunoprecipitation. The latter were also implemented by the combinations of sst2A with other GPCRs. The existence of co-precipitated heterodimers was shown in the immunoblot in all cases. The heterodimers sst2A-sst4 and sst2A-MOR1 were examined for changes in function caused by the dimerisation. The agonist induced endocytosis (internalisation) was proven by immunocytochemistry and confocal microscopy. The activation of mitogen activated kinases ERK1 and ERK2, was shown by immunoblot. All of the receptors were localised (when singularly expressed) or co-localised (when co-expressed) on the plasma membrane, resepctively. But, the sst2A-sst4 internalised and activated the ERK1, and the ERK2 remained unchanged when compared to the sst2A and sst4 cell lines. When sst2A and MOR1 were co-expressed, MOR1 was also internalized remained unchanged after treatment with MOR1 selective DAMGO [D-Ala2,Me-Phe4,Gly5-ol]-enkephalin). However, after stimulation with the sst2A selective ligands (L-779,976),both receptors were internalized together. Furthermore, both receptors became desensitised after a four hour preincubation with L-779,976 or DAMGO, and could not be further stimulated by their own agonists. The experiments show that heterodimerisation is a common phenomenon, but does not always lead to a modulation in the receptor properties. It is possible that all GPCRs form long, ologomeric double chains in the plasma membrane, which could cause the various physiological consequences. It is therefore probable, that there are more mechanisms specific to cell type, which regulate heterodimerisation and its consequences.