Cytochalasins and colchicine increase the lateral mobility of human chorionic gonadotropin-occupied luteinizing hormone receptors on ovine luteal cells

Abstract

To determine whether cytoskeletal proteins restrict the mobility of LH-receptor complexes occupied by hCG, we measured the rate of lateral diffusion and the fraction of mobile receptor-hormone complexes by fluorescence photobleaching recovery on luteal cells treated with microtubule and microfilament disruptors and on portions of the plasma membrane separated from the underlying cytoskeleton (blebs). Enzymatically dispersed ovine luteal cells obtained on day 11 from superovulated ewes were pretreated for 1 h at 37 C with cytochalasin B (20 micrograms/ml), cytochalasin D (20 micrograms/ml), or colchicine (40 micrograms/ml). These drugs had no effect on the lateral diffusion of cell membrane glycoproteins labeled with tetramethylrhodamine isothiocyanate-derivatized succinyl Concanavalin-A. However, drug treatment caused a significant increase in the diffusion coefficient and mobile fraction of hCG-occupied LH receptors compared to values obtained on untreated cells. hCG-occupied LH receptors on untreated cells exhibited a mobile fraction of 10.3%, and these mobile complexes had a diffusion coefficient of 3.3 X 10(-11) cm2 sec-1.hCG-occupied LH receptors on cytochalasin B-treated cells had a diffusion coefficient of 9.0 X 10(-11) cm2 sec-1, with 26% fluorescence recovery. Cytochalasin D and colchicine caused significantly greater increases in hCG mobility to 22.0 and 22.5 X 10(-11) cm2 sec-1, respectively, although the mobile fraction remained at about 25-30%. That binding of hCG to the LH receptor restricts lateral mobility through interactions of the hormone-occupied receptor complex with the cytoskeleton is further indicated by results obtained on membrane blebs. The fraction of mobile hCG-occupied LH receptors on blebs was 52.3%, and complexes had a diffusion coefficient of 1.7 X 10(-10) cm2 sec-1. The hormone-induced restriction of LH receptor lateral diffusion had no effect on the lateral mobility of tetramethylrhodamine isothiocyanate-epidermal growth factor-labeled receptors.