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Days After Implant figure 1-33 Regulation of receptor performance, the Kd (A), and receptor concentration via either up-regulation (B) or down-regulation (C). In A, three saturation curves for a receptor are shown; the Kd's for binding curves K, L, and M are 10~10, 10"9, and 10~8 M, respectively. If the prevailing hormone concentration bathing the target cell is 10"® M, then depending upon the affinity of the receptor for its ligand, the level of receptor occupancy can vary from 80% (curve K) to 50% (curve L) to 20% (curve M). In B, homologous down-regulation of the insulin receptor is displayed; cultured lymphocytes were exposed to various concentrations of hormone, and total insulin receptor levels were determined. Gavin, J. R., Roth, J., Neville, D. M., De Meyts, P., and Buelle, D. N. (1974). Insulin-dependent regulation of insulin receptor concentration: A direct demonstration in cell culture. Proc. Natl. Acad. Sci. USA 71 (84-88). In C homologous up-regulation of the prolactin receptor (PRL) is displayed. Hypophysectomized rats were implanted with pituitaries under the kidney capsule. The increase of the hormone in serum preceded the increase of the receptor in liver. (Posner, B. I., Kelly, P. A., and Friesen, H. G. (1975). Prolactin receptors in rat liver: Possible induction by prolactin. Science 188, 57-59.

free hormone and measuring the amount bound with some convenient assay. The half-maximal value of a plot of receptor-bound hormone (ordinate) versus total free hormone concentration (abscissa) approximates the dissociation constant, which will have a specific hormone concentration in molarity as its value. Hormone bound to receptor is corrected for nonspecific binding of the hormone to membranes. This can be measured conveniently if the hormone is radiolabeled by measuring receptor plus labeled hormone (radioactive) and receptor plus radioactive hormone after the addition of an excess (100-1000 times) of unlabeled hormone. The excess unlabeled hormone will displace the high-affinity hormone-binding sites but not the low-affinity nonspecific binding sites. Thus, when the "radioactive plus nonradioactive" curve is subtracted from the "radioactive" curve (Figure 1-30), an intermediate curve will represent specific binding to receptor. This is of critical importance when the receptor is measured in a system containing other proteins. As an approximation, 20 times the Kd value of hormone is usually enough to saturate the receptor.

Most measurements of Kd are made by using Scatch-ard analysis, which is a manipulation of the equilibrium equation. The equation can be developed by a number of routes, but can be envisioned from mass

TABLE 1-6 Membrane-Bound Receptor Hormone Transduction Systems

Hormones acting via utilization of a cAMP system cAMP decrease cAMP increase

Hormones acting via a cGMP system

Hormones acting via IPj pathway

Hormones acting via ion channels

Bradykinin

Norepinephrine (a2-adrenergic) Somatostatin

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