Functional interaction of prostaglandin E receptor EP 3 subtype with guanine nucleotide-binding proteins, showing low-affinity ligand binding

Abstract

The functional interaction of prostaglandin E (PGE) receptor EP 3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP 3 receptor cDNA-transfected Chinese hamster ovary cells. PGE 2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP 3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE 2 binding to the membranes was increased by GTPγS, and PT treatment also increased the binding activity to the same level as that increased by GTPγS, but the sensitivity of GTPγS was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE 2 binding activity with the following preference: Gi1 = Gi2 > Gi3 > Go , but GTPγS completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in K d without the change of B max , and due to suppression of association rate. [ 3H]PGE 2-bound EP 3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTPγS was eluted at the position of M r = approx . 60 kDa, similar to the relative molecular mass of EP 3 receptor deduced from its amino acid sequence. In contrast, [ 3H]PGE 2-bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M r = approx . 130 kDa, corresponding to the M r of the complex of EP 3 receptor and Gi2, but GTPγS shifted the position of its elution from M r = 130 to 60 kDa. Furthermore, addition of PGE 2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE 2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi1 = Gi2 > Gi3 > Go . These results indicate that EP 3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.