| Abstract: | Two steroid binding states of an estrogen receptor each with different equilibrium constants (Kd values) Rx (Kd = 0.06 nM) and Ry (Kd = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third nonestrogen binding form of the receptor, designated Rnb, is now described which exists in short-term estrogen withdrawn chick oviduct cytosol. A model is presented in which the receptor can be interconverted between the three states. The interconversion is monitored by Scatchard analysis, sucrose density gradient analysis, and affinity labeling using [3H]tamoxifen aziridine followed by receptor purification with estrogen receptor monoclonal antibody affinity chromatography and sodium dodecyl sulfate-gel electrophoresis. The results are consistent with each state existing in different conformations having a common molecular weight of approximately 66,000. This paper defines the conditions and nucleotide requirements for the Rnb to Ry conversion. The conversion to the steroid binding form is induced by ATP, ADP, and GTP. Cyclic nucleotides are ineffective. There is a specific requirement for Mg2+; neither Ca2+ nor Mn2+ will substitute. Nonhydrolyzable nucleotide analogues were tested for their relative efficiency to convert Rnb to Ry. Conversion occurred with alpha,beta-methylene adenosine triphosphate, but beta,gamma-methylene adenosine triphosphate and alpha,beta-methylene adenosine diphosphate were inert. Thus, activation of Rnb to form Ry appears to be catalyzed by an event requiring the loss of the terminal phosphoryl moiety from either ATP or ADP. Receptor derived from conversion of Rnb to Ry has the same physical properties as native Ry. Activation of Rnb is to Ry specifically; no increase in the Rx form of estrogen receptor was ever observed. The accompanying paper similarly describes the Rx to Ry conversion. Since these data also explain observations made with glucocorticoid and with epidermal growth factor receptors, it is speculated that the receptor interconversion model may have general application to hormone action. |
