Cheryl S. Watson University o/Texas Medical Branch Cellular steroid action has been thoroughly studied in the nuclear compartment. However, nuclear steroid receptor mechanisms have been unable to explain some of the rapid activities of steroids, partiCUlarly those which occur in a time frame of seconds to minutes [reviewed in (1;2)]. Based on these and other considerations, an alternative membrane-associated receptor form was long ago proposed to exist (3). Others interpret the location of the steroid receptors mediating these rapid effects as peri membrane or cytoplasmic. New experimental tools have been brought to bear on the topic of receptors for steroids which mediate non-genomic actions, and thus investigative activity and focus regarding this type of steroid receptor has recently increased significantly. However, there may be multiple answers to the question "how do steroids mediate rapid nongenomic effects?" Steroid actions initiated at the cell membrane can impinge on important phases in the lifespan of a cell: proliferation, migration, differentiation, and release of hormones or neurotransmitters functioning as signals to other cells.

In addition to the well-characterized genomic mechanism of steroid action that uses intracellular receptors, steroid hormones also signal through nongenomic processes that use membrane receptors. A membrane receptor for corticosterone (CORT) has been described in brains of the roughskin newt (Taricha granulosa). This receptor is believed to be a G-protein coupled receptor because corticosterone binding is inhibited by guanyl nucleotides and enhanced by Mg2. The studies described in this thesis use biochemical, pharmacological and molecular techniques to characterize the newt neuronal membrane glucocorticoid receptor (mGR) in pursuit of its molecular identification. The mGR was successfully solubilized from newt neuronal membranes and conditions were defined that maintained corticosterone binding activity for further study. The solubilized receptor was partially purified using standard chromatographic techniques and an immobilized ligand affinity resin (CORT-Sepharose). These chromatographic studies were combined with the use of a novel photoaffinity ligand (azido-CORT) to biochemically characterize the mGR protein, finding that it is an acidic glycoprotein with an apparent molecular weight of 63 kDa and an isoelectric point of approximately 5.0. Pharmacological studies with mGR showed that a subset of kappa opioid ligands displaced corticosterone from the receptor binding site with K[subscript i] values in the nanomolar to low-micromolar range. The interaction of mGR with kappa opioid ligands was specific because no mu-, delta-, or orphanin-specific opioid ligands were effective at displacing corticosterone from the receptor. These data suggest that the newt neuronal mGR may be a kappa-opioid like receptor. Finally, molecular studies were used to clone a novel newt brain protein, neuronal axonal protein 22 (NAP-22), that was identified in a protein differential display strategy designed to identify mGR. Studies with the cloned and expressed NAP-22 protein suggest that it is not the mGR but, instead, may be a mGR-associated protein. These studies provided new information about the biochemical and pharmacological properties of mGR, and may have discovered a protein that is associated with the newt neuronal mGR.

During the last two decades, progress in steroid hormone research has resulted in the development of new approaches to contraception as well as diagnosis and treatment of endocrine disorders and cancers. Although significant advances have been made in the purification, characterization, immunochemistry and molecular biology of steroid receptors, the precise molecular mechanism of steroid hormone action has remained obscure. This book captures the detailed presentations made at the first conference on Steroid Receptors in Health and Disease held at Meadow Brook Hall, Oakland University in the fall of 1987. The purpose of this international con ference was to facilitate scientific exchange toward a better understand ing of the mode of action of steroid hormones. The scientific sessions consisted of poster presentations and state-of-the-art lectures, the latter of which make up this volume. The first chapter is meant to provide the reader with a more general background of the topics covered in the book, as well as to discuss certain theme-related issues that are either not yet well-established or accepted or are in the stage of infancy. It is hoped that this volume will serve as a useful treatise for students and investigators interested in basic and clinical aspects of biological regulation by steroid hormones. A task of this magnitude could not have been undertaken without the encouragement, advice and continued generous assistance of the members of the scientific committee. I am gratefully indebted to Drs.

The past few years have witnessed the emergence of steroid hormones as the wonder molecules which generate as much discussion in the scientific literature as they do in a typical living room. This transition has been a result of the tremendous public and scientific interest in the normal functioning of the hor mones as well their suggested involvement in several clinical conditions. In the recent past, notable scientific and technological advances have been made in the areas of contraception and regulation of fertility. Steroid receptors are the indis pensable mediators of hormonal responses and are complex protein molecules which appear to exist in association with other, yet undefined, proteins and/or factors. Receptors for vitamin D, retinoic acid and the thyroid hormones share structural similarities with steroid receptors, and the roster of this superfamily is still expanding. While our knowledge of the diversity and magnitude of steroid effects has advanced, the precise mode of steroid hormone action has alluded investigators. This volume brings together an international team of prominent investigators who discuss their most recent work on the basic and clinical aspects of steroid/nuclear receptors. The contributions represent updated versions of the invited presentations made at The Second Meadow Brook Conference on Steroid Receptors in Health and Disease. I am grateful to my colleagues on the Scientific Committee: Etienne Baulieu, Jack Gorski, Benita Katzenellenbogen, David Toft and James WittJiff, who provided the vision and guidance in formulating an out standing program.

The papers in this volume were presented at the Symposium on Steroid Hormone Receptor Systems held October 18-20, 1978, at the Worcester Foundation for Experimental Biology, Shrewsbury, Mass. The meeting was organized to review, discuss, and disseminate new knowledge about the regulation and function of the receptor proteins which mediate estrogen, progestin, glucocorticoid, and androgen action. The symposium brought together leading scientists whose interests span the spectrum of biological organization. On this occasion, Drs. Elwood V. Jensen and Etienne E. Baulieu were honored as recipients of the Tenth Annual Gregory Pincus l1emorial Award for their pioneering studies of steroid hormone receptors. The material covered in this book focuses on the molecular mechanisms which control receptor site availability and function. The topics discussed include recent work on receptor antibodies, antiestrogen action, receptor heterogeneity, nuclear binding and processing of receptor, receptor activation and inactivation mechan isms, interactions between receptor systems, influence of biological rhythms, membrane binding sites, and the evolution of steroid-bind ing proteins. Although studies on steroid receptors have led to the development of new approaches for cancer therapy and contraception, much remains to be discovered in this rapidly expanding field. Hope fully, this book will provide added impetus to the quest for a full understanding of steroid receptor systems by drawing attention to the unresolved questions yet to be answered. Hhile the book is intended primarily for those interested in hormone action, it should be of value to a more general audience including cell, molecular, and developmental biologists.

A distinguished team of principal investigators and their associates describe in step-by-step detail a cross-section of the latest research techniques available for studying the endocrine system. As a basis for sophisticated biochemical analysis of receptor properties, the contributors provide methods for the production and purification of a variety of receptors, including progesterone, glucocorticoid, and androgen. Other protocols allow the reader to experiment with DNA binding characteristics, hormone binding assays, and the use of combinatorial chemistry for drug discovery. A series of novel methods utilizing the latest advances in immunochemistry, yeast two-hybrid screening, and fluorescence are included for the detection and analysis of a variety of cellular proteins that influence steroid receptor effectiveness.