Functional cDNA clones for two isoforms of the mouse prostaglandin E receptor EP3 subtype derived from alternative RNA splicing were obtained. The two isoforms are only different in the sequence of the putative cytoplasmic carboxyl-terminal tail and their hydrophobicity; one isoform, named EP3alpha, has a hydrophilic tail, and the other, named EP3beta, has a hydrophobic tail. When expressed, the two receptors displayed identical ligand binding properties but different responses to guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Without a change in the B(max) value, GTPgammaS increased K(d) for prostaglandin E2 of EP3beta and decreased that of EP3alpha. These effects were abolished by the treatment of membranes with pertussis toxin and restored by the addition of G(i2). Although both isoforms exerted inhibition of forskolin-induced cAMP accumulation, three orders lower concentrations of agonists were required for EP3alpha than EP3beta for 50% inhibition of cAMP formation. A similar difference in agonist potency was observed also for agonist-induced stimulation of GTPase activity in membranes. Thus, the two receptors with different carboxyl-terminal tails show different coupling to the G(i) protein, leading to the opposite responses to GTP in the ligand binding affinity and to different affinities of the agonist-occupied receptors to the G proteins.