Estimating the rate of externalization of gonadotrophin-releasing hormone receptors in ovine anterior pituitary cells in vitro.
We report an estimate of the rate of externalization of unstimulated receptors for gonadotrophin-releasing hormone (GnRH), and derive from this the turnover time of the unstimulated receptor. The binding of the GnRH antagonist [acetyl-D-pCl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]-GnRH to dispersed sheep anterior pituitary cells was non-saturable at 37 degrees C. Further experiments showed that the binding had two distinct phases. We suggest that these phases correspond to the initial, saturable binding to existing plasma membrane receptors, followed by binding to receptors as they are inserted into the surface membrane. The two processes are temporally distinct, and can be inhibited independently by pharmacological manipulations. The initial phase was inhibited by treatments that could be expected to reduce the number of active receptors on the cell surface (preincubation of the cells for 30 min with 100 micrograms neuraminidase/ml or 50 mumol GnRH/ml), and was complete in less than 30 min after the addition of the antagonist tracer. The second phase occurred continuously in the presence of tracer, and was reduced or abolished by inhibitors of microtubule function (100 mumol vinblastine/l), protein synthesis (25 micrograms cycloheximide/ml), or energy metabolism (0.25 mmol 2,4-dinitrophenol/l). The rate of insertion of receptors into the plasma membrane was calculated from the rate of increase of the second phase of binding. The calculated rate implies a 100% turnover of unstimulated receptors every 150 min. In contrast, previously published estimates of the rate of internalization of the GnRH-receptor complex in the rat pituitary suggest that the stimulated receptor is turned over much faster.
Starling
L
L
Department of Obstetrics and Gynaecology, Wellington School of Medicine, University of Otago, New Zealand.
McIntosh
J E
JE
McIntosh
R P
RP
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011966:Receptors, LHRH / Q000378:metabolism*; D012756:Sheep; D013997:Time Factors
D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; C043124:N-Ac-(4-Cl-Phe)(1)-(4-Cl-Phe)(2)-Trp(3)-Lys(6)-AlaNH2(10)-LHRH; D000242:Cyclic AMP