Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized mu-opioid receptors from rat brain: Coimmunoprecipitation of the G proteins G(alpha o), G(alpha i1), and G(alpha i3)
E. Chalecka-franaszek et al., Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized mu-opioid receptors from rat brain: Coimmunoprecipitation of the G proteins G(alpha o), G(alpha i1), and G(alpha i3), J NEUROCHEM, 74(3), 2000, pp. 1068-1078
Antibodies directed against the C-terminal and the N-terminal regions of th
e mu-opioid receptor were generated to identify the G proteins that coimmun
oprecipitate with the mu receptor. Two fusion proteins were constructed: On
e contained the 50 G-terminal amino acids of the mu receptor, and the other
contained 61 amino acids near the N terminus of the receptor. Antisera dir
ected against both fusion proteins were capable of immunoprecipitating simi
lar to 70% of solubilized rat brain mu receptors as determined by [H-3][D-A
la(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin ([H-3]DAMGO) saturation binding. Th
e material immunoprecipitated with both of the antisera was recognized as a
broad band with a molecular mass between 60 and 75 kDa when screened in a
western blot. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) had an EC50
of 0.4 nM in diminishing [H-3]DAMGO binding to the immunoprecipitated pell
et. The ratio of G proteins to mu receptors in the immunoprecipitated mater
ial was 1:1. When the material immunoprecipitated with affinity-purified an
tibody was screened for the presence of G protein ac subunits, it was deter
mined that G(alpha o), G(alpha i1), G(alpha i3), and to a lesser extent G(a
lpha i2), but not G(alpha s) or G(alpha q/11), were coimmunoprecipitated wi
th the CL receptor. Inclusion of GTP gamma S during the immunoprecipitation
process abolished the coimmunoprecipitation of G proteins.