Identification and Characterization of an Abundant Phosphoprotein Specific to the Large Luteal Cell*

Abstract

An abundant protein with a relative mol wt of 32K present specifically in the large cells of the pregnant rat corpus luteum has been identified. Separation of large and small luteal cells by elutriation, followed by protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), have revealed that the 32K protein was present as a major protein in the large luteal cells but was practically absent in the small cell population. This protein appears to be highly tissue and cell specific and resolves into three protein species by two-dimensional SDS-PAGE with the major protein having an isoelectric point (pI) greater than or equal to 8.5. It was not detected in preantral follicles or placentas of the same pregnant rats, or in any other tissue examined. After subcellular fractionation, the 32K protein(s) was found in the particulate fraction and was localized principally in the microsomal compartment. Autoradiographic analysis of 35S-amino acid-labeled tissue demonstrated that the 32K protein(s) is synthesized in the corpus luteum. When particulate fractions from small and large cells were incubated in the presence of [gamma-32P]ATP followed by SDS-PAGE, phosphorylation of the 32K protein was apparent. Phosphorylation of this protein was not enhanced by the addition of cofactors for cAMP, Ca2(+)-calmodulin- or Ca2(+)-phospholipid-dependent kinases. Experimental inhibition of steroidogenesis with amino-glutethimide caused a remarkable reduction in the luteal content of this 32K protein whereas estradiol and human CG treatment increased its content. In summary, we have discovered and partially characterized a unique 32K protein(s) which is expressed and phosphorylated only in large luteal cells of the corpus luteum. This protein(s), which is regulated by estradiol formed locally, may serve as a powerful marker for both the large luteal cell and estrogen action in the corpus luteum.