T cells are considered to be unresponsive to testosterone due to the absence of androgen receptors (AR). Here, we demonstrate the testosterone responsiveness of murine splenic T cells in vitro as well as the presence of unconventional cell surface receptors for testosterone and classical intracellular AR. Binding sites for testosterone on the surface of both CD4(+) and CD8(+) subsets of T cells are directly revealed with the impeded ligand testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Binding of the plasma membrane impermeable testosterone-BSA conjugate induces a rapid rise (<5 s) in [Ca2+]i of Fura-2-loaded T cells. This rise reflects influx of extracellular Ca2+ through non-voltage-gated and Ni2+-blockable Ca2+ channels of the plasma membrane. The testosterone-BSA-induced Ca2+ import is not affected by cyproterone, a blocker of the AR. In addition, AR are not detectable on the surface of intact T cells when using anti-AR antibodies directed against the amino and carboxy terminus of the AR, although T cells contain AR, as revealed by reverse transcription-polymerase chain reactions and Western blotting. AR can be visualized with the anti-AR antibodies in the cytoplasm of permeabilized T cells by using CLSM, though AR are not detectable in cytosol fractions when using the charcoal binding assay with 3H-R1881 as ligand. Cytoplasmic AR do not translocate to the nucleus of T cells in the presence of testosterone, in contrast to cytoplasmic AR in human cancer LNCaP cells. These findings suggest that the classical AR present in splenic T cells are not active in the genomic pathway. By contrast, the cell surface receptors for testosterone are in a functionally active state, enabling T cells a nongenomic response to testosterone.

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca(2+)], which is due to release of Ca(2+) from intracellular Ca(2+) stores. This Ca(2+) mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).

We have shown that estrogens and calcitriol, the hormonally active form of vitamin D, increase the concentration of intracellular calcium ([Ca 2؉ ] i ) within 5 s by mobilizing calcium from the endoplasmic reticulum and the formation of inositol 1,4,5-trisphosphate and diacylglycerol. Because the activation of effectors as phospholipase C (PLC) coupled to G-proteins is the early event in the signal transduction pathway leading to the inositol 1,4,5-trisphosphate formation and to [Ca 2؉ ] i increase, we described different PLC isoforms (␤1, ␤2, ␥1, and ␥2, but not ␤4) in female rat osteoblasts using Western immunoblotting. The data showed that phospholipase C ␤ was involved in the mobilization of Ca 2؉ from the endoplasmic reticulum of Fura-2-loaded confluent osteoblasts by calcitriol and 17␤ estradiol, and PLC ␥ was ineffective. The data also showed that only a PLC ␤1 linked to a Pertussis toxin-insensitive G-protein and a PLC ␤2 coupled to a Pertussis toxin-sensitive G-protein are involved in the effects of calcitriol and 17␤ estradiol on the mobilization of Ca 2؉ from intracellular Ca 2؉ stores. In conclusion, these results may be an important step toward understanding membrane effects of these steroids and may be an additional argument in favor of membrane receptors to steroid hormones.An increase in the turnover of inositol lipids in response to receptor is one of the most important molecular mechanisms used by cells for transmembrane signaling. The initial event is the hydrolysis of phosphatidylinositol 4,5-bisphosphate, a reaction catalyzed by a phosphoinositide-specific phospholipase C (PLC), 1 which generates two intracellular second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (1-4). Inositol 1,4,5-trisphosphate binds to specific receptors on the endoplasmic reticulum (5) and mobilizes intracellular calcium, whereas diacylglycerol activates protein kinase C (6), which results in increased phosphorylation of cellular proteins.Molecular cloning has revealed at least three major families of PLC, ␤, ␥, and ␦ (7-9). Each of these families occurs in a number of isoforms. The enzymes are classified on the basis of their size and their immunological and structural similarities. The PLC isoforms have two highly conserved domains, X and Y, which form the active site of the protein. PLC ␦ and PLC ␥ proteins differ from PLC ␤ in that they have shorter C-terminal extensions past the end of the Y domain (9). This diversity among the PLC isoforms also extends to distinct mechanisms of regulation and function for the three PLC families. PLC ␥ is regulated via the phosphorylation of tyrosine residues between the X and Y domains by receptor tyrosine kinases (10 -12). PLC ␤ enzymes, of which there are four isoforms, PLC ␤1-4, are regulated via heterotrimeric G-proteins in response to an agonist binding to a receptor (13-15). The way in which PLC ␥ is regulated is not yet known, but enzyme activity is not affected by either the G-protein subunits or by receptor tyrosine kinases (16).The activat...

The Fura-2 method is used to examine a possible action of 17L L-estradiol (E P ) on [ influx and Ca P+ release from intracellular stores is also inducible by plasma membrane impermeable E P conjugated to BSA. E P -BSA-FITC binds to the surface of T cells of both the CD4 + and CD8 + subset. Our data suggest a novel E P -signalling pathway in T cells which is not mediated through the classical nuclear estrogen receptor response but rather through putative plasma membrane receptors for E P .z 1998 Federation of European Biochemical Societies.

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