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Mol Biol Cell. Oct 1999; 10(10): 3113–3123.
PMCID: PMC25566

Testosterone Signaling through Internalizable Surface Receptors in Androgen Receptor-free Macrophages

Keith R. Yamamoto, Monitoring Editor

Abstract

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 [Ca2+], which is due to release of Ca2+ from intracellular Ca2+ stores. This Ca2+ 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.

INTRODUCTION

Steroid hormones act on target cells through their cognate receptors belonging to the intracellular steroid receptor superfamily (reviewed by Evans, 1988 blue right-pointing triangle; Beato, 1989 blue right-pointing triangle; Jensen, 1996 blue right-pointing triangle). These are hormone-regulated transcription factors eliciting either induction or repression of specific genes (reviewed by Kumar and Tindall, 1998 blue right-pointing triangle). Evidence, however, is accumulating that steroids can also cause nongenomic responses of cells, i.e., responses not mediated through classical nuclear receptors but rather responses initiated at the plasma membrane, presumably through unconventional surface receptors (reviewed by Brann et al., 1995 blue right-pointing triangle; Wehling, 1997 blue right-pointing triangle; Grazzini et al., 1998 blue right-pointing triangle; Nadal et al., 1998 blue right-pointing triangle; Nemere and Farach-Carson, 1998 blue right-pointing triangle).

Testosterone, for example, is a steroid hormone that has been described to exert both genomic effects and, recently, also nongenomic effects. Genomic responses of testosterone are mediated through intracellular androgen receptors (ARs), which are 110-kDa proteins with domains for androgen binding, nuclear localization, dimerization, DNA binding, and transactivation (reviewed by Zhou et al., 1994 blue right-pointing triangle; Quigley et al., 1995 blue right-pointing triangle). The nongenomic effects are assumed to be mediated through unconventional receptors in plasma membranes. In rat osteoblasts, these membrane receptors have been recently shown to belong to the class of membrane receptors coupled to phospholipase C via a pertussis toxin-sensitive G-protein, which, after binding of testosterone, mediate a rapid increase in intracellular free [Ca2+] ([Ca2+]i) and inositol 1,4,5-trisphosphate formation (Lieberherr and Grosse, 1994 blue right-pointing triangle).

However, unconventional testosterone receptors in plasma membranes can be suspected to be classical intracellular ARs tightly associated with the plasma membrane. This view is not unlikely, because ARs, in contrast to most other steroid receptors, are reported to be localized in the cytoplasm and not in nuclei (Simental et al., 1991 blue right-pointing triangle; Zhou et al., 1994 blue right-pointing triangle). Also, rat osteoblasts are known to be typical testosterone target cells containing intracellular AR (Colvard et al., 1989 blue right-pointing triangle). Here, however, we provide evidence for functional testosterone receptors in plasma membranes of macrophages, which lack classical intracellular AR and which respond to plasma membrane-impermeable testosterone nongenomically with intracellular Ca2+ mobilization.

MATERIALS AND METHODS

IC-21 Cells

The SV40-transformed peritoneal macrophage cell line IC-21 (ATCC no. TIB-186) generated from a C57BL/6 mouse was obtained from the American Type Culture Collection (Manassas, VA). The cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) and l-glutamine (Life Technologies, Eggenstein, Germany) supplemented with 10% FCS, 50 μM β-mercaptoethanol, and 3.024 g of NaHCO3 at 37°C, 5% CO2, and 96% humidity, subcultured once per week, and incubated for 24 h in serum-free medium before use.

RNA Isolation

RNA was isolated from IC-21 cells and testes removed from C57BL/10 mice using the GTC-CsCl method (Sambrook et al., 1989 blue right-pointing triangle)

Reverse Transcription (RT)-PCR

The PCRs were carried out with the RNA PCR kit from Perkin Elmer (Weiterstadt, Germany). The initial random-primed RT was performed with 1 μg of total RNA in a Minicycler (MJ Research, Biozym, Oldendorf, Germany) and AmpliTaq DNA Polymerase (Perkin Elmer), and four different oligonucleotide primer pairs were used for PCR amplification of the AR. The primer pair AR-P1 (5′-GACCTTGGATGGAGAACTACTCCG-3′) and AR-M1 (5′-GGTTGGTTGTTGTCATGTCCGGC-3′) spanned 511 nucleotides (nt) of the DNA-binding domain of AR. The carboxyl terminus of the AR was probed with three different primer pairs: AR-P2 (5′-ACGTCCTGGAAGCCATTGAGCC-3′) and AR-M2 (5′-CTTGGTGAGCTGGTAGAAGCGC-3′), as well as the sense primers AR-P3 (5′-GAATGTCAGCCTATCTTTCTTAACG-3′) and AR-P4 (5′-TCCTTTGCTGCCTTGTTATCTAGC-3′) together with the antisense primer AR-M3 (5′-TGCCTCATCCTCACACACTGGC-3′). As a control for the integrity of the RNA isolated from IC-21 cells, the low abundant mRNA of the mzfm gene was amplified by RT-PCR using the primer pairs mzfm-P1 (5′-GGCTTAACACCCGAGAGTTCC-3′) and mzfm-M1 (5′-TTATCCTGAGCTGACTGAGGG-3′) as well as mzfm-P2 (5′-GGGTCTATCGCCTGCATCAAGG-3′) and mzfm-M2 (5′-TCCTCACTCTCATGGCTCGG-3′) (Wrehlke et al., 1997 blue right-pointing triangle, 1999 blue right-pointing triangle). The AR-P1 and AR-M1 were subjected to 32 cycles at 94°C for 1 min, at 56°C for 1 min, and at 72°C for 1 min. The other primer pairs were used in 32 cycles at 95°C for 1 min, at 60°C for 1 min, and at 72°C for 1 min. PCR fragments were separated in 2% Tris borate-EDTA gels, eluted, and cloned into the pMOSBlue T vector (Amersham, Braunschweig, Germany).

DNA Sequencing

Clones were sequenced with Thermo Sequenase fluorescent-labeled sequencing kit (Amersham) and analyzed with the LICOR sequencer (MWG, Ebersberg, Germany)

Western Blotting

Proteins were separated in 8% SDS polyacrylamide slab gels (Laemmli, 1970 blue right-pointing triangle) and blotted onto nitrocellulose membranes (0.45 μm pore size; Schleicher & Schuell, Dassel, Germany) with a Biometra (Göttingen, Germany) semidry blot cell. Membranes were incubated with the anti-AR antibody AR (N-20) (Santa Cruz Biotechnology, Heidelberg, Germany) at a concentration of 0.1 μg/ml diluted in 10 mM Tris-HCl, pH 7.5, 0.15 mM NaCl, and 0.05% Tween (TST) at 23°C for 1 h, washed three times with TST for 10 min, and incubated at 23°C for 1 h with HRP-conjugated goat-anti-rabbit immunoglobulin G (IgG; Santa Cruz Biotechnology) diluted 1:50,000 in TST. Antibody detection was performed by the enhanced chemiluminescence plus Western blotting detection system (Amersham).

Determination of [Ca2+]i

IC-21 cells in IMDM were grown on poly-l-lysine-coated glass coverslips until confluence. Then they were washed twice with 20 mM HEPES buffer, pH 7.2, supplemented with 130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 1 mM Na2HPO4, and 1 mg/ml glucose before they were loaded with 1 μM Fura-2/AM (Amersham, Les Ulis, France) in the same HEPES buffer at room temperature for 30 min. The Ca2+ measurements were performed in a Hitachi (Mountain View, CA) F-2000 spectrofluorometer at 37°C. Reagents were added directly to the cuvette under continuous stirring. Testosterone, testosterone 3-(O-carboxymethyl)oxime-BSA (testosterone-BSA), estradiol, flutamide, and pertussis toxin were from Sigma (St. Quentin, Fallavier, France); 1-dehydrotestosterone was from Steraloids (Wilton, NH). Testosterone-BSA contained <0.1% free testosterone. Cyproterone was kindly provided by Schering (Berlin, Germany); U-73122 [1-(6-((17β-3-metoxyestra-1,3,5(10)-trien-17-yl)-amino)hexyl)-1H-pyrrole-2,5-dione] and U-73343 [1-(6-((17β-3-metoxystra-1,3,5(10)-trien-17-yl)-amino)-hexyl-2,5-pyrrolidine-2,5-dione] were from Biomol Reseach Laboratory (Plymouth, MA). Hormones were dissolved in ethanol. The final concentration of ethanol in the cuvette never exceeded 0.01%, which did not affect [Ca2+]i (cf. Lieberherr and Grosse, 1994 blue right-pointing triangle). The Fura-2 fluorescence was measured at 340 nm (calcium-bound Fura-2) and 380 nm (free Fura-2) for excitation and 510 nm for emission. The [Ca2+]i was computed from the ratio of 340:380 nm fluorescence values as desribed previously (Grynkiewicz et al., 1985 blue right-pointing triangle).

Labeling with Testosterone-BSA-FITC

IC-21 cells (5 × 106 cells/ml) in IMDM were allowed to adhere onto poly-l-lysine-coated glass coverslips overnight, washed twice with PBS+ (140 mM NaCl, 2.7 mM KCl, 6.4 mM Na2HPO4, 1.4 mM KH2PO4, 0.5 mM MgCl2, 0.9 mM CaCl2, pH 7.2), and then incubated at room temperature for 5 s up to 1 h with 100 μl of 1.5 × 10−5 M testosterone-BSA-FITC (Sigma, Deisenhofen, Germany). Only BSA-FITC and BSA were used in the corresponding control experiments. After two washings with PBS+, cells were fixed with 0.5% paraformaldehyde for 30 min. Coverslips were briefly rinsed with PBS+ and mounted on slides in a 1:1 (vol/vol) mixture of glycerol and Vectashield (Vector Laboratories, Burlingame, CA) containing 2% (wt/vol) 1,4-diazabicyclo-[2.2.2]octane (Merck, Darmstadt, Germany).

Confocal Laser Scanning Microscopy

The specimens were analyzed with a Leica TCS NT confocal laser scanning microscope (CLSM), version 1.5.451 (Leica Lasertechnik, Heidelberg, Germany). FITC fluorescence was excited by a 488-nm argon laser line, and Cy3 and TRITC fluorescence was excited by a 568-nm krypton laser line, respectively. Z-series optical sections were taken at 0.5-μm intervals (Benten et al., 1998 blue right-pointing triangle, 1999 blue right-pointing triangle) and evaluated using Adobe Photoshop 5.0 for Windows (Adobe Systems, Mountain View, CA) and CorelDRAW 8 for windows (Corel, Ottawa, Ontario, Canada).

Flow Cytometry

IC-21 cells (107 cells/ml) were suspended in PBS+, and aliquots of 150 μl were centrifuged. The cell pellets were labeled with testosterone-BSA-FITC, BSA-FITC, and the anti-AR antibody AR (N-20) (2 μg/ml) for 5 s up to 1 h as described above. Labeling with rabbit antiserum to sex hormone-binding globulin (SHBG) (batch 64.5; a gift from W. Rosner, St. Luke’s-Roosevelt Hospital Center, New York, NY) was performed with dilutions between 1:60,000 and 1:60 for 1 h. Anti-rabbit IgG (whole molecule) FITC conjugate (working dilution, 1:320; Sigma) was used as secondary antibody for 45 min as described previously (Benten et al., 1991 blue right-pointing triangle). Cells were analyzed in a FACScan (Becton Dickinson, Sunnyvale, CA) with a sample size of 10,000 cells gated on the basis of forward and side scatter. The data were stored and processed using the FACScan software as described previously (Benten et al., 1991 blue right-pointing triangle).

Internalization of Testosterone-BSA-FITC

Intact IC-21 cells were incubated at room temperature or 37°C for 15 min or 1 h with testosterone-BSA-FITC (1.5 × 10-5 M), BSA-FITC (1.5 × 10-5 M), concanavalin A (Con A)-rhodamine (1:50; Vector), or a rat anti-mouse F4/80 antibody (2 μg/ml; a gift from H. Mossmann, Max-Planck-Institut for Immunobiology, Freiburg, Germany) as first antibody, Biotin-SP-conjugated AffiniPure mouse anti-rat IgG (heavy and light chain; 1:500; Jackson ImmunoResearch, West Grove, PA) as secondary antibody, and streptavidin-fluorescein (6 μg/107 cells; Amersham). Colocalization was performed with LysoTracker Red DND-99 (10 μM; Molecular Probes, Göttingen, Germany), the anti-clathrin antibody heavy chain (N-19) (2 μg/ml; Santa Cruz Biotechnology), and a secondary donkey anti-goat-Cy3 antibody (1:200; a gift from P. Traub, Max-Planck-Institut for Cell Biology, Ladenburg, Germany) or with the anti-caveolin antibody caveolin-1 (N-20) (2 μg/ml; Santa Cruz Biotechnology) and as secondary antibody TRITC-conjugated AffiniPure goat anti-rabbit IgG (heavy and light chain; 1:80; Jackson ImmunoResearch). The samples were fixed, embedded, and analyzed by CLSM as described above.

Perturbation of Internalization

Intact IC-21 cells were preincubated at different temperatures or at 37°C with different substances for varying periods before incubation with testosterone-BSA-FITC (1.5 × 10−5 M, if not otherwise stated). The substances were NaN3 (Merck), pertussis toxin (Sigma), U-73122, U-73343, and cytochalasin B and nocodazole (Sigma). The samples were fixed and analyzed by flow cytometry and CLSM as described above.

RESULTS

Absence of Intracellular AR

Different techniques were used to examine the presence of classical intracellular AR in mouse macrophages of the cell line IC-21. Both intact cells and permeabilized cells were investigated by flow cytometry using the anti-AR antibody AR (N-20), which is directed against an epitope corresponding to the amino acids 2–21 mapping at the amino terminus of the AR. Incubation of intact IC-21 cells with this antibody did not result in any significant labeling of the cells (Figure (Figure1A).1A). After permeabilization of IC-21 cells, incubation with AR (N-20) resulted in a slight increase in fluorescence intensity. However, this fluorescence was not AR specific, because it could not be competitively displaced by an AR (N-20)-specific blocking peptide (Figure (Figure1A).1A). Also, the anti-AR antibody AR (N-20) did not detect AR in IC-21 cells in Western blots, although this antibody reacted with the AR band at 110 kDa in AR-expressing human prostate cancer LNCaP cells (Figure (Figure1B)1B) (Taplin et al., 1995 blue right-pointing triangle). Moreover, RT-PCR was used to detect AR mRNA in IC-21 cells and in mouse testes as a control. Using primers spanning the DNA-binding domain and three different regions from the carboxyl terminus of the AR, RT-PCR revealed the expected bands in testes but not in IC-21 macrophages (Figure (Figure1C).1C). DNA sequencing confirmed that the PCR fragments derived from testes RNA contained the predicted regions of the AR. Moreover, the RNA isolated from IC-21 cells was intact, because the low abundant mRNA of the single-copy gene mzfm (Wrehlke et al., 1997 blue right-pointing triangle, 1999 blue right-pointing triangle) could be amplified by the same RT-PCR procedure using two different primer pairs (Figure (Figure1C).1C).

Figure 1
Absence of intracellular AR in IC-21 cells. (A) Flow cytometry of intact and permeabilized IC-21 cells incubated for 1 h with the anti-AR antibody AR (N-20) and secondary fluorescent antibody (Sec. Ab-FITC). In permeabilized cells, the blocking peptide ...

Testosterone-induced Ca2+ Mobilization

IC-21 cells were loaded with Fura-2 to determine the effect of testosterone on [Ca2+]i. Testosterone at the physiological concentration of 10 nM triggered an immediate spike in [Ca2+]i, which represented a Ca2+ increase by ~100 nM (Figure (Figure2A).2A). Such a spike was also induced when the cells were preincubated with the AR blockers cyproterone and flutamide in excess (Figure (Figure2B).2B). The Ca2+ increase may be due to influx of extracellular Ca2+ and/or release of Ca2+ from intracellular Ca2+ stores. To test this, extracellular Ca2+ was removed by EGTA before testosterone was added. Testosterone was still able to induce the Ca2+ spike (Figure (Figure2C).2C). However, the testosterone-induced Ca2+ spike was totally abolished by the direct phospholipase C inhibitor U-73122 but not by the inactive control compound U-73343 (Figure (Figure2D).2D). Also, the Ca2+ spike could be inhibited by pertussis toxin (Figure (Figure2E).2E). Mn2+ did not induce any quenching after testosterone treatment (Figure (Figure2F).2F).

Figure 2
Testosterone-induced Ca2+ mobilization in IC-21 cells. (A) Testosterone causes an immediate Ca2+ spike. (B) Cells were treated with cyproterone for 30 min or flutamide for 60 min before adding testosterone. (C) Cells were incubated with ...

However, when testosterone was added for a second or third time shortly after the first addition, it induced a prolonged elevation of [Ca2+]i instead of a Ca2+ spike (Figure (Figure3A).3A). This prolonged elevation in [Ca2+]i was due to both Ca2+ release and Ca2+ import, because after removal of extracellular Ca2+, treatment with testosterone resulted only in a Ca2+ spike instead of a prolonged elevation (Figure (Figure3B).3B).

Figure 3
Ca2+ Responses of IC-21 cells to testosterone and testosterone-BSA. (A) A second or third addition of testosterone shortly after the first treatment induces a sustained increase in [Ca2+]i rather than a Ca2+ spike. ...

Testosterone coupled to BSA, which is not freely permeable through the cell membrane, had the same effects on [Ca2+]i as free testosterone. It induced first a Ca2+ spike, whereas a second addition caused a prolonged elevation of [Ca2+]i (Figure (Figure3C).3C). The Ca2+ spike was only due to Ca2+ release, whereas the prolonged elevation of [Ca2+]i was due to both Ca2+ release and Ca2+ import (Figure (Figure3,3, D and E). Moreover, pertussis toxin blocked the testosterone-BSA-induced mobilization of intracellular Ca2+ (Figure (Figure33F).

The amount of released Ca2+ induced by the first addition of both testosterone and testosterone-BSA increased with increasing concentrations, reaching apparent saturation at ~10 nM testosterone and 100 nM testosterone-BSA, respectively (Figure (Figure4A).4A). Moreover, cells responded to a second addition of testosterone again with a Ca2+ spike when the period between first and second additions exceeded at least 10 min (Figure (Figure4B).4B).

Figure 4
Effect of different concentrations of testosterone, testosterone-BSA, and testosterone pretreatment on [Ca2+]i of IC-21 cells. (A) Increase in [Ca2+]i with increasing concentrations of testosterone and testosterone-BSA, ...

The rapid nongenomic effects of testosterone on [Ca2+]i of IC-21 cells were specific for testosterone. First, estradiol caused Ca2+ responses differing from those induced by testosterone (Figure (Figure5).5). Thus, estradiol at a concentration of only 1 nM induced a Ca2+ spike of 100 nM Ca2+, whereas the Ca2+ spike was halved to 50 nM when estradiol was added at 10 nM (Figure (Figure5A).5A). Moreover, a second addition of estradiol resulted in a second Ca2+ spike instead of a prolonged elevation. In addition, the estradiol-induced Ca2+ spikes were due to both Ca2+ release from intracellular stores and Ca2+ influx, because removal of extracellular Ca2+ by EGTA led to a shortened and reduced Ca2+ spike after estradiol treatment (Figure (Figure5B).5B). Second, 1-dehydrotestosterone, the structure of which is very similar to that of testosterone, did not induce any specific Ca2+ response of the cells (Figure (Figure5C).5C).

Figure 5
Effect of estradiol and 1-dehydrotestosterone on [Ca2+]i of IC-21 cells. (A) Treatment of cells with 1 nM estradiol resulted in a higher increase in [Ca2+]i than 10 nM estradiol. A second addition of estradiol ...

Surface Binding of Testosterone

Testosterone binding sites were identified on the surface of intact IC-21 cells. When cells were incubated with the impeded ligand testosterone-BSA coupled to FITC for 5 s, flow cytometry revealed an increase in fluorescence intensity (Figure (Figure6A).6A). Incubation with BSA-FITC alone or together with free testosterone did not result in any significant labeling in comparison with unlabeled control cells (cf. Figure Figure7A).7A). CLSM detected the fluorescence of the bound testosterone-BSA-FITC exclusively on the surface of IC-21 cells (Figure (Figure6B).6B). The same labeling pattern on the surface showed the plasma membrane marker ConA-rhodamine (cf. Figure Figure7B).7B). There is some evidence that the SHBG can bind to specific receptors on the plasma membranes, which are able to mediate rapid effects of testosterone and estradiol (Rosner et al., 1998 blue right-pointing triangle). However, the surface of IC-21 cells had not bound any significant amounts of SHBG, as identified by flow cytometry and CLSM using an anti-SHBG antibody (our unpublished data).

Figure 6
Surface binding sites of testosterone and their internalization in intact IC-21 cells. (A) Cells were incubated with testosterone-BSA-FITC for various periods between 5 s and 1 h and then analyzed by flow cytometry. (B) CLSM of cells incubated with testosterone-BSA-FITC ...
Figure 7
Selective internalization of surface binding sites of testosterone. (A) Flow cytometry of IC-21 cells treated with the indicated substances for 15 min. (B) CLSM of cells incubated for 15 min with the indicated substances and the corresponding FITC-labeled ...

Selective Internalization of Testosterone Receptors

There is evidence that G-protein-coupled surface receptors can be sequestered (reviewed by Koenig and Edwardson, 1997 blue right-pointing triangle). To identify such a possible sequestration of surface testosterone receptors, IC-21 cells were incubated with testosterone-BSA-FITC between 5 s and 1 h and analyzed by flow cytometry and CLSM. Flow cytometry revealed an increased labeling with progressive incubation periods (Figure (Figure6A).6A). When incubation lasted for 5 s or 1 min, >80% of the cells were labeled with testosterone-BSA-FITC. After 5 min, however, the percentage of labeled cells was increased to >95%, and the fluorescence intensity of the cells was higher. Thereafter, the number of fluorescent cells remained about the same, whereas the fluorescence intensity of the cells still increased with progressing incubation times, reaching a maximum after ~1 h. Obviously, cells bound increasing amounts of testosterone-BSA-FITC with progressing incubation times. In parallel with the increase in fluorescence intensity, CLSM revealed an increasing punctate fluorescence inside cells (Figure (Figure6B).6B). Whereas the fluorescence was exclusively localized on the cell surface after 5 s and 1 min, punctate weak fluorescence emerged after 5 min inside cells at their periphery, besides surface fluorescence. After 15 min and 1 h, the punctate fluorescence was increased in intensity and was distributed throughout the whole cytoplasm inside cells.

The internalization of testosterone binding sites was selective. BSA alone or BSA-FITC did not induce any sequestration (Figure (Figure7A).7A). Also, when cells were incubated with free testosterone together with BSA-FITC for 15 min, there was no sequestration, although sequestration was observed when cells were incubated in parallel with testosterone-BSA-FITC (Figure (Figure7,7, A and B). Moreover, internalization occurred neither with surface-bound ConA-rhodamine nor with the macrophage specific surface marker F4/80 identified by a rat monoclonal antibody against F4/80. Even if the surface labeling of IC-21 cells was performed in the presence of testosterone, there was no internalization of ConA-rhodamine and F4/80 (Figure (Figure77B).

Gross Characteristics of Receptor Internalization

The sequestrated testosterone-BSA-FITC was not contained in acidic vesicles. The latter were identified by CLSM using LysoTracker Red DND-99. The vesicles stained with LysoTracker Red DND-99 did not colocalize with the green punctate fluorescence of testosterone-BSA-FITC (Figure (Figure8A).8A). Also, the sequestrated testosterone binding sites did colocalize neither with clathrin as detected by anti-clathrin antibodies (Figure (Figure8B)8B) nor with caveolin as monitored by anti-caveolin antibodies (Figure (Figure8C).8C).

Figure 8
CLSM colocalization of the sequestered surface binding sites of testosterone. (A) IC-21 cells were incubated in parallel with testosterone-BSA-FITC and LysoTracker Red DND-99 at 37°C for 1 h. Testosterone-BSA-FITC did not colocalize with acidic ...

To determine the effect of various parameters on receptor internalization, intact IC-21 cells were incubated with testosterone-BSA-FITC for 15 min, and subsequently fluorescence intensity was analyzed by flow cytometry, and fluorescence localization was analyzed by CLSM. Figure Figure9A9A shows that internalization of testosterone-BSA-FITC could be competitively reduced by testosterone but not by the structurally similar compound 1-dehydrotestosterone. Moreover, internalization of surface-bound testosterone-BSA-FITC was largely inhibited at temperatures below ~16°C, whereas temperature did not affect binding of testosterone-BSA-FITC to the cell surface (Figure (Figure9B).9B). Depletion of ATP by sodium azide resulted in a decrease of fluorescence intensity by ~40% (Table (Table1).1). This fluorescence was localized almost exclusively on the cell surface. Internalization of surface-bound testosterone-BSA-FITC also could be abolished by preincubation with pertussis toxin (Table (Table1).1). The phospholipase C inhibitor U-73122 but not the inactive compound U-73343 also blocked internalization, because preincubation with 2 μM U-73122 for 2 min resulted in complete surface localization of testosterone-BSA-FITC, whereas controls have internalized testosterone-BSA-FITC, as revealed by the ~10% higher fluorescence intensity (Table (Table1).1). Finally, internalization obviously involved cytoskeletal elements. Both the tubulin blocker nocodazole and the microfilament blocker cytochalasin B inhibited internalization but not surface binding of testosterone-BSA-FITC (Table (Table1).1). Control cells revealed higher fluorescence intensities by ~25% because of internalization of surface-bound testosterone-BSA-FITC.

Figure 9
Flow cytometry of the internalization of surface-bound testosterone-BSA-FITC in IC-21 cells. (A) Cells were incubated for 15 min with testosterone-BSA-FITC (10−6 M) in the absence (Control) or in the presence of a 10-fold excess of unlabeled testosterone ...
Table 1
Effect of various substances on sequestration of surface-bound testosterone-BSA-FITC

DISCUSSION

This study provides evidence for the presence of functional receptors for testosterone on the surface of the intracellular AR-free macrophages of the cell line IC-21. Using the impeded ligand testosterone-BSA-FITC, which is not freely permeable to the plasma membrane, CLSM localizes testosterone binding sites on plasma membranes of intact IC-21 cells. These membrane receptors for testosterone cannot be identical with classical intracellular ARs, because ARs are not expressed in IC-21 cells. ARs were detectable by neither flow cytometry nor Western blotting using the anti-AR antibody AR (N-20) directed against the amino terminus of AR or at the mRNA level by RT-PCR using primers probing the DNA-binding domain and three different regions of the carboxyl terminus of the AR. Moreover, the AR blockers cyproterone and flutamide were not able to inhibit binding of testosterone to IC-21 cells. In accordance, previous studies also could not identify intracellular AR in macrophages of the cell line RAW 264.7 (Benten et al., 1999 blue right-pointing triangle) and in macrophages of different tissues (Gulshan et al., 1990 blue right-pointing triangle; Frazier-Jessen and Kovacs, 1995 blue right-pointing triangle; Miller et al., 1996 blue right-pointing triangle), although the presence of AR was reported in immature monocytic cells (Danel et al., 1985 blue right-pointing triangle; Cutolo et al., 1993 blue right-pointing triangle). The reason for this discrepancy is unknown, but the expression of AR in monocytes and macrophages may be developmentally regulated, as it is postulated, for example, to occur in T cells (Kovacs and Olsen, 1987 blue right-pointing triangle; Viselli et al., 1995 blue right-pointing triangle; Benten et al., 1999 blue right-pointing triangle).

The testosterone receptors on the surface of IC-21 cells are functionally coupled to intracellular Ca2+ homeostasis. Indeed, binding of the plasma membrane-impermeable testosterone-BSA induces a rapid increase in [Ca2+]i. This increase, which is also induced by the physiological concentration of 10 nM testosterone, occurs within seconds and is predominantly due to the release of Ca2+ from intracellular Ca2+ stores. However, external Ca2+ also contributes to the increase in [Ca2+]i, which is imported through Ca2+ channels, becoming particularly evident upon a second stimulus with testosterone or testosterone-BSA. Moreover, the specificity of membrane testosterone receptors is further corroborated by our findings 1) that the testosterone-induced Ca2+ release is saturable, 2) that 1-dehydrotestosterone, which is very similar in structure to testosterone, is largely inactive to induce specific Ca2+ release, and 3) that estradiol evokes Ca2+-responses differing from those of testosterone. Moreover, our data reveal that the membrane testosterone receptors not only are functionally coupled with Ca2+ channels in the plasma membrane but also belong to that class of membrane receptors that are coupled to phospholipase C via a pertussis toxin-sensitive G-protein, because Ca2+ release can be blocked by both pertussis toxin and the phospholipase C inhibitor U-73122 but not by the inactive compound U-73343.

The G-protein-coupled receptors for testosterone (GPCRT) in IC-21 cells exhibit a novel peculiarity, i.e., agonist-triggered sequestration. Indeed, this sequestration manifests itself as internalized punctate fluorescence of testosterone-BSA-FITC. The internalization begins a short while 1) after binding of testosterone-BSA-FITC to the surface of IC-21 cells and 2) after Ca2+ mobilization by testosterone. The latter, therefore, may be a precondition for ligand-induced internalization of the GPCRT. This view is also supported by the fact that the phospholipase C inhibitor U-73122 and pertussis toxin inhibit both Ca2+ mobilization and internalization of surface-bound testosterone-BSA-FITC. Our data reveal that the internalization process is not a simple fluid endocytosis or a constitutive endocytotic pathway of IC-21 cells but rather is ligand specific. Thus, internalization of surface-bound testosterone-BSA-FITC is competitively inhibited by testosterone but not by 1-dehydrotestosterone. In addition, GPCRT internalization is selective; i.e., only distinct plasma membrane domains are internalized excluding surface markers such as F4/80 and Con A-rhodamine. GPCRT internalization is consistent with other findings showing that a wide variety of G-protein coupled receptors (GPCRs), e.g., the prototypic β2-andrenergic receptor and angiotensin II type 1A receptor, become sequestrated after ligand binding (von Zastrow and Kobilka, 1992 blue right-pointing triangle; Moore et al., 1995 blue right-pointing triangle; Koenig and Edwardson, 1997 blue right-pointing triangle), which is considered important for regulation of signaling, recycling, down-regulation, and responsiveness of the GPCRs (Yu et al., 1993 blue right-pointing triangle; Pippig et al., 1995 blue right-pointing triangle; Koenig and Edwardson, 1997 blue right-pointing triangle).

In general, GPCRs internalize via the clathrin-coated vesicle-mediated endocytotic pathway (Doxsey et al., 1987 blue right-pointing triangle; Robinson et al., 1996 blue right-pointing triangle; Zhang et al., 1996 blue right-pointing triangle), although entry also may be mediated via caveolae (Chun et al., 1994 blue right-pointing triangle; Kiss and Geuze, 1997 blue right-pointing triangle). However, our data suggest that GPCRT internalization does not proceed along such pathways. Indeed, the punctate fluorescence of internalized testosterone-BSA-FITC in IC-21 cells is associated with neither clathrin nor caveolin nor acidic vesicles. Obviously, the ligand-triggered entry of GPCRT into IC-21 cells is mediated by a clathrin- and caveolin-independent internalization pathway (cf. Roettger et al., 1995 blue right-pointing triangle; Robinson et al., 1996 blue right-pointing triangle). On the other hand, the internalization process of GPCRT in IC-21 cells resembles that observed for numerous other GPRCs insofar as this process is critically dependent on temperature, ATP, and cytoskeletal elements (von Zastrow and Kobilka, 1994 blue right-pointing triangle; Roettger et al., 1995 blue right-pointing triangle; Morrison et al., 1996 blue right-pointing triangle; Koenig and Edwardson, 1997 blue right-pointing triangle; Koenig et al., 1997 blue right-pointing triangle).

Testosterone signaling through testosterone surface receptors has also been described in rat osteoblasts (Lieberherr and Grosse, 1994 blue right-pointing triangle) and murine T cells (Benten et al., 1997 blue right-pointing triangle, 1999 blue right-pointing triangle). Howerver, there exist differences in comparison with IC-21 cells. For instance, plasma membranes of rat osteoblasts also possess GPCRT, but these do not become sequestrated upon agonist stimulation and mediate both Ca2+ import of external Ca2+ via voltage-gated Ca2+ channels and Ca2+ release from intracellular Ca2+ stores (Lieberherr and Grosse, 1994 blue right-pointing triangle). In T cells, the membrane testosterone receptors are not sequestrable, and they mediate only ligand-induced Ca2+ import through non–voltage-gated, Ni2+-blockable Ca2+ channels (Benten et al., 1997 blue right-pointing triangle, 1999 blue right-pointing triangle). At present it is too premature to discriminate whether the membrane testosterone receptors in all these different cell types are different or identical but coupled to different signaling pathways dependent on the cell type. In this context, there is also information available that not all cells possess membrane testosterone receptors. For instance, hepatocytes do not respond to testosterone with Ca2+ mobilization, although the cells are able to mobilize Ca2+ in response to progesterone and estradiol (Sanchez-Bueno et al., 1991 blue right-pointing triangle).

Collectively, our data unequivocally show the presence of functional unconventional GPCRT in plasma membranes of IC-21 cells, which do not mediate the classical genomic AR response but rather initiate novel nongenomic testosterone signaling pathways involving Ca2+ as one of several other possible intracellular mediators. Signal integration into cell functioning and the physiological significance remain to be determined. In particular, it remains elusive whether the testosterone-induced increase in [Ca2+]i per se modulates secondarily expression of specific genes, for example, through Ca2+-responsive promotor elements, negative Ca2+-responsive promotor elements, and/or Ca2+-modulatable transcription factors such as NF-AT, NF-κB, and c-Jun N-terminal kinase (Negulescu et al., 1994 blue right-pointing triangle; Dolmetsch et al., 1997 blue right-pointing triangle).

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