Differences in the lateral mobility of receptors for luteinizing hormone (LH) in the luteal cell plasma membrane when occupied by ovine LH versus human chorionic gonadotropin

Abstract

Receptors for LH are internalized by ovine luteal cells 50 times slower when occupied by hCG than when occupied by ovine LH (oLH). To determine if differences in the rate of internalization were due to differences in the lateral mobility of the hormone-receptor complexes in the cell membrane, the diffusion coefficients of oLH- and hCG-LH receptor complexes were measured using fluorescence photobleaching recovery methods. Tetramethylrhodamine isothiocyanate (TRITC)-labeled oLH and hCG, which retained full ability to bind to receptor, were bound to LH receptors on enzymatically dispersed ovine luteal cells. Molecules labeled with TRITC within a 3-micron 2 region of the cell surface were bleached by a 500-msec pulse of 3 mW laser light at a wavelength of 514.5 nm. The laser beam intensity was then attenuated 20,000-fold, and fluorescence from the bleached area was measured by single photon counting as unbleached fluorescent hormone-receptor complexes diffused into the region. Data were analyzed on-line by a NOVA 3/12 computer. The oLH-LH receptor complex had a diffusion coefficient of 1.9 +/- 1.0 X 10(-10) cm2/sec-1, a value comparable to that of cell surface proteins nonspecifically labeled with succinylated Concanavalin A. Fluorescence recovery after photobleaching was 35%. In contrast, hCG-LH receptor complexes were immobile on the time scale of the experiment, implying that the diffusion coefficient was substantially less than 1 X 10(-11) cm2/sec-1. Deglycosylated hCG-TRITC bound to LH receptor had a diffusion coefficient (1.1 +/- 0.1 X 10(-10) cm2/sec-1) similar to that of receptors occupied by oLH. Thus, it appears that the carbohydrate portion of the hCG molecule plays a role in decreasing the mobility of the receptor for LH. These data demonstrate that the rate of lateral movement of the LH receptor in the plasma membrane of luteal cells appears to be modulated by the nature of the bound hormone.