The orientation of the lutropin/choriogonadotropin receptor in rat luteal cells as revealed by site-specific antibodies.

Polyclonal antibodies were raised against peptides corresponding to specific regions of the rat luteal lutropin/choriogonadotropin (LH/CG) receptor. One of these (anti-LHR02) was directed against a 13 amino acid sequence that occurs within the large N-terminal hydrophilic domain, and another (anti-LHR06) was directed against the 14 most C-terminal amino acids of the receptor (as deduced from the open reading frame of the cDNA). These antisera, which we show to be highly specific for the LH/CG receptor, were used in indirect immunofluorescence experiments with primary cultures of rat luteal cells to determine the orientation of this receptor within the plasma membrane. Anti-LHR02 was shown to specifically bind to intact cells. In contrast, anti-LHR06 did not bind to intact cells. When the cells were first permeabilized with detergent and then incubated with this antibody, however, an intense and specific immunofluorescence was observed. These studies clearly show that the orientation of the LH/CG receptor in the plasma membrane is such that the N-terminal domain is located extracellularly, while the C terminus is located intracellularly. Thus, we have experimentally confirmed that the orientation of this receptor as predicted from the amino acid sequence is indeed correct.