Functional interaction of prostaglandin E receptor EP3 subtype with guanine nucleotide-binding proteins, showing low-affinity ligand binding.

The functional interaction of prostaglandin E (PGE) receptor EP3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP3 receptor cDNA-transfected Chinese hamster ovary cells. PGE2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE2 binding to the membranes was increased by GTP gamma S, and PT treatment also increased the binding activity to the same level as that increased by GTP gamma S, but the sensitivity of GTP gamma S was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE2 binding activity with the following preference: Gi1 = Gi2 > Gi3 > GO, but GTP gamma S completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in Kd without the change of Bmax, and due to suppression of association rate. [3H]PGE2-bound EP3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTP gamma S was eluted at the position of M(r) = approx. 60 kDa, similar to the relative molecular mass of EP3 receptor deduced from its amino acid sequence. In contrast, [3H]PGE2-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 EP3 receptor and Gi2, but GTP gamma S shifted the position of its elution from M(r) = 130 to 60 kDa. Furthermore, addition of PGE2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi1 = Gi2 > Gi3 > GO. These results indicate that EP3 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.