Introduction

There is abundant but circumstantial evidence indicating that brain cells possess putative membrane steroid receptors (for review, see refs. 1 and 2). It seems appropriate to emphasize once more the several criteria that need to be fulfilled before accepting the concept that steroid hormones bind to specific sites in cellular membranes of the nervous system (CNS), representing protein molecules with the properties of receptors.

The receptors should have high affinity because physiological hormone concentrations are usually in the namolar range, though in different physiological states (3-5), a broad range of fluctuation in blood levels does exist. Alternately, the local effective concentration of the hormones in a particular region of the CNS may not represent accurately the blood concentration (6-8). They should have also high specificity, so that closely related hormones will still preferentially bind to their cognate receptors and remain functionally distinct. There should be a finite number of receptors in any given membrane preparation so they should become saturable with increased doses of the ligand. The kinetics of the receptor-ligand interaction should follow the laws of mass action, with the ligand binding the receptor in a reversible reaction reaching equilibrium rapidly, at which stage the rate of association equals the rate of dissociation. However,

From: Contemporary Endocrinology: Neurosteroids: A New Regulatory Function in the Nervous System Edited by: E.-E. Baulieu, P. Robel, and M. Schumacher © Humana Press Inc., Totowa, NJ

under certain condition the hormone-receptor (HR) complex may bind to an effector (X) with a Kd that favors the formation of the trimeric complex (HR:X), complicating the kinetics of a bimolecular reaction (9,10). The receptors should have a tissue distribution appropriate to the action of the hormone; that is, it should be present in the target organs of a particular steroid and absent from the tissue unresponsive to the steroid. Within a particular organ, such as the brain, for instance, the receptor may have a selective distribution in the cell membrane of the neurons or glia and/or in specific compartments of such cells, as it is the case for the mitochondria that bind estradiol (E) through oligomycin sensitivity conferring protein (OSCP), a subunit of the ATP synthase (11,12). The receptor should be an integral membrane protein that can be recovered after detergent solubi-lization and inactivated by proteolytic enzymes. Within limits, the specific binding should increase linearly with the increase in the number of receptors, i.e., concentration of proteins in the membrane preparation. Eventually, and most importantly, the activation of the receptor by the ligand should be coupled to a biological response. Ultimately, the membrane steroid receptors should be cloned and molecularly characterized.

Though there are strong supporting evidence for specific membrane sites for several steroid hormones in the CNS (1,2,13), in this chapter we describe some of the aforementioned criteria above as applied to one of those membrane steroid receptors, the putative membrane estrogen receptor (mER), as an example of these new series of membrane receptors. Thus, in what follows, and using the estradiol-bovine serum albumin conjugate (E-6-BSA) as a probe for the putative mER, we address the following questions:

1. Is the E-6-BSA conjugate biologically active?

2. Does the conjugate specifically bind to subcellular brain preparations?

3. Are these membrane estradiol binding sites novel or physiologically relevant proteins?

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