Technical Reference #1782

1782.

Direct Interaction of GABA-B Receptors with M2 Muscarinic Receptors Enhances Muscarinic Signaling Stephanie B. Boyer; Sinead M. Clancy; Miho Terunuma; Raquel Revilla-Sanchez; Steven M. Thomas; Stephen J. Moss; Paul A. Slesinger, The Salk Institute for Biological Studies, Journal of Neuroscience, 29(1782), (2009)
Link To Paper

Materials & Methods:
Molecular biology and tissue culture. The following constructs were used: GIRK2c expressed in pcDNA3.1( ) human M1 muscarinic receptor expressed in peCFP-N1 and human -opioid receptor ( OR) expressed in peCFP-N1. TheM2 muscarinic receptor was fused to cyan fluorescence protein (CFP) (M2R-CFP) as described previously (Clancy et al. 2007). GBR1/GBR2-yellow fluorescent protein (YFP) and GBR1/GBR2-CFP were created as described previously (Fowler et al. 2007). GBR2 3-YFP was created by engineering a SacII site into GABABR2 after R747 and subcloning into peYFP-N1 using HindIII/SacII. GBR2 1 and 2 were made by engineering an AgeI site at amino acids 820 and 776 respectively and subcloning into peYFP-N1 using EcoRI/AgeI. GBR1R2-YFP was created by engineering a KpnI site into a conserved region of GBR1 and GBR2 substituting a phenylalanine for a lysine (R1F883L/R2F739L) and subcloning the C terminus of GBR2 into GBR1-YFP using KpnI/ AgeI. These substitutions resulted in receptors that functioned similar to wild type. GBR2R575D-YFP was created using the QuikChange XL kit from Stratagene. Chimeras swapping the C-terminal domains between M1 andM2 muscarinic receptors were created by overlap PCR (Finley et al. 2004). Glutathione S-transferase (GST) fusion constructs were created by fusing the region of interest to the 3 end of GST using pGEX-2T. H8-fusion constructs were created using pHis8.3. Fusion constructs were expressed in BL21 ) Escherichia coli and affinity purified as previously described (Lunn et al. 2007).

Microscopic Technique
TIRF Microscopy

Cell Type(s)
Neuronal PC12 cells