ISOLATION AND CHARACTERIZATION OF A cDNA FROM ATLANTIC CROAKER (Micropogonias undulatus) OVARIES ENCODING A NOVEL PROTEIN WITH THE CHARACTERISTICS OF A MEMBRANE ANDROGEN RECEPTOR

Abstract

There is now extensive evidence that in addition to the wellcharacterized genomic actions mediated through nuclear receptors, steroids can also exert “nonclassical” rapid actions initiated at the cellsurface. However, the identities of the receptors that mediate many of these nonclassical steroid actions remain unresolved. Both nuclear receptors and novel receptors for estrogens and progestins have been identified in the plasma membranes of a variety of target cells. In contrast, there is a relative lack of information on the identities of membrane receptors mediating nonclassical androgen actions. In the present study, a protein displaying androgen binding characteristics typical of androgen membrane receptors was partially purified from the ovaries of Atlantic croaker. The purified protein was used to produce polyclonal antibodies that were subsequently used to screen an ovarian cDNA library. Positive clones were sequenced and a novel cDNA encoding a protein of approximately 40 kDa with several putative transmembrane domains was identified. Structural analyses revealed no sequence similarities on nucleotide or amino acid levels between this novel gene and nuclear steroid receptors or steroid membrane receptors. The novel cDNA was stably transfected into the breast cancer cell line SKBR-3 to examine androgen binding characteristics, expression patterns and cellsignaling. Western blot and immunocytochemical analyses of transfected cells showed that the recombinant protein had a molecular weight of approximately 40 kDa and was located in the plasma membranes. Testosterone (T) binding to plasma membranes prepared from transfected cells was increased 303 ± 87% compared to that in untransfected cells. Saturation and Scatchard analyses indicated the presence of a single, high affinity (Kd – 12.75 ± 3.22 nM), low-capacity (Bmax – 2.8 ± 0.5 pmol/mg protein) testosterone binding site on the membranes of transfected cells. The specific testosterone binding to the membrane fraction was readily displaceable with rapid association and dissociation kinetics (t½ – 1.5 ± 0.4 and 2.2 ± 0.7 minutes respectively). Steroid binding to the recombinant protein was specific for androgens. Progesterone displayed low affinity, and 17-beta estradiol (E2) and cortisol showed no affinity for the putative receptor in competitive binding assays. Highest binding affinity was observed with T, but other androgens such as 5alpha-dihydrotestosterone (DHT), androstenedione and 11-ketoandrostenedione also displayed significant binding affinities. Interestingly, the teleost androgen, 11- ketotestosterone, showed no binding affinity for the recombinant protein. Western blot and RT-PCR analyses of croaker tissues showed expression of the putative androgen membrane receptor protein and mRNA in reproductive tissues as well as in the brain and liver, whereas there was little or no expression in heart, gills, or intestine. Moreover, the expression of the novel protein in oocyte membranes varied during the ovarian cycle and was upregulated by gonadotropin and steroid treatments in vitro, suggesting it has a physiological role in croaker reproduction. Taken together, the results are consistent with the suggestion that the cDNA and protein discovered in Atlantic croaker ovaries is a novel androgen membrane receptor, unrelated to any known steroid receptors. (poster)