Immunoexpression of Tyro 3 Family ReceptorsTyro 3, Axl, and Merand Their Ligand Gas6 in Postnatal Developing Mouse Testis
Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Correspondence to: Daishu Han, PhD, Department of Cell Biology, PUMC & CAMS 5 Dong Dan San Tiao, Beijing 100005, P.R. China. E-mail: daishu{at}public.bta.net.cn
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(J Histochem Cytochem 53:13551364, 2005)
Key Words: Tyro 3 family receptors Gas6 immunohistochemistry Sertoli cell Leydig cell testis mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tyro 3 family receptors are widely expressed in adult tissues such as neural, lymphoid, vascular, smooth muscular, reproductive tissue, and retina, and in primary and tumor cell lines derived from these sources (Graham et al. 1995; Crosier et al. 1997
; Prieto et al. 2000
; Konishi et al. 2004
; Valverde et al. 2004
). It has been shown (Lu et al. 1999
; Lu and Lemke 2001
) that triple mutant (Tyro3/Axl/Mer/) mice displayed multiple major organ defects, neurological abnormalities, severe lymphoproliferative disorders, spermatogenesis failure, and physiological deficits, suggesting that Tyro 3 family play significant biological roles in multiple tissues. How they perform the function is unknown.
More attention deserves to be paid to the functions of Tyro 3 family receptors on mammalian spermatogenesis. The triple knockout male mice completely lose the production of mature sperm, whereas single- and double-gene mutant male animals were fertile (Lu et al. 1999). These findings suggest that these three receptors are essential regulators of spermatogenesis and that their functions in gonadal development are compensated by each other. However, the expression patterns of Tyro 3 family receptor and their ligand Gas6 have not been extensively studied. Although the mRNAs of Tyro 3, Axl, Mer, and Gas6 in testicular cells have been examined by in situ hybridization and Northern blot in a few studies (Lu et al. 1999
; Chan et al. 2000
; Wong and Lee 2002
), the results have not been consistent. A study on the immunoexpression of Tyro 3 family receptors and Gas6 in their protein levels would be helpful to clear up this matter. Furthermore, a precise localization and expression kinetics of Tyro 3, Axl, Mer, and Gas6 proteins in testis would be a basis to further study their functions in spermatogenesis.
In this study, immunohistochemistry staining was used to examine the immunoexpression patterns of receptor Tyro 3, Axl, Mer, and ligand Gas6 in postnatal developing testis.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
Goat anti-Tyro 3, Axl, and Mer polyclonal antibodies and rabbit anti-Gas6 polyclonal antibody were used for detecting protein Tyro 3, Axl, Mer, and Gas 6. All primary antibodies and reagents related to immunohistochemistry staining and Western blotting were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), except where noted otherwise.
Western Blotting
Testis from d35 mouse was lysed on ice with lysis buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, protease inhibitors: 1 mU/ml aprotinin, 0.1 mM leupeptin, 0.5 mM PMSF) for 1 hr. Insoluble materials were removed by centrifugation at 12,000 x g at 4C for 15 min; supernatant was mixed with loading buffer and boiled for 5 min. Then the samples were loaded on 10% SDS-PAGE and subsequently electrotransferred onto poly(vinylidene difluoride) transfer membranes (Millipore; Bedford, MA). After blocking with 2% BSA in PBS containing 0.1% Tween-20 (PBS-T) for 1 hr, the electrotransferred membranes were incubated with anti-Tyro 3, anti-Axl, anti-Mer, or anti-Gas6 antibody at 1:1000 dilution at 4C overnight. After washing with PBS-T, the membrane was incubated with horseradish peroxidaseconjugated rabbit anti-goat IgG (for Tyro 3, Axl, and Mer) or goat anti-rabbit IgG (for Gas6) antibody (Zhongshan; Beijing, China) at room temperature for 1 hr. After washing with PBS-T, antigen-antibody complex was visualized by using an enhanced chemiluminescence detection kit (Zhongshan).
Tissue Section Preparations
Postnatal day 3 (d3), d7, d14, d21, d35, and d56 normal mice were anesthetized with CO2 and then killed by cervical dislocation. The testes were immediately removed and fixed with 4% paraformaldehyde in 0.1 M PBS, pH 7.4, for 12 hr at room temperature. The samples then were dehydrated in ethanol, cleared with xylene, embedded in paraffin, sectioned at 5 µm thickness in a microtome, mounted onto poly-L-lysine precoated glass slides, and dried overnight at 37C for immunohistochemistry staining.
Immunohistochemistry Staining
Immunohistochemistry staining was conducted by the avidin-biotin-peroxidase complex method. Samples were deparaffinized in xylene at room temperature and rehydrated with a graded ethanol and then with distilled water. Next, they were incubated in PBS containing 3% H2O2 for 15 min to inactive endogenous peroxidase activity. After a brief wash in PBS, they were soaked in a citrate buffer and microwaved at 100C for 10 min for antigen retrieval. Before addition of the primary antibodies, the samples were incubated with normal rabbit serum for Tyro 3, Axl, and Mer or normal goat serum for Gas6 to block nonspecific binding. They were then incubated overnight with the primary antibody; goat anti-Tyro 3, Axl, and Mer antibodies; and rabbit anti-Gas6 antibody in a moist chamber at 4C. This was followed by three 5-min washes in PBS. Next, the samples were incubated with the biotinylated rabbit anti-goat IgG for Tyro 3, Axl, and Mer or goat anti-rabbit IgG for Gas6 for 30 min and then with streptavidin-peroxidase complex for 30 min. After three 5-min washes in PBS, the peroxidase-binding sites of all the samples were demonstrated by the diaminobenzidine method. All sections were counterstained with hematoxylin and mounted with Canada balsam (Sigma; St Louis, MO) for observation after dehydrated in ethanol and clarification with xylene. The primary antibodies were used at 1:400 dilutions for Tyro 3 and Axl or 1:800 for Mer and Gas6. Negative control sections were incubated with preimmune goat serum or rabbit serum instead of primary antibody, or with primary antibody preabsorbed with blocking peptide.
Isolation of Sertoli Cells and Germ Cells
The procedure for the isolation of Sertoli cells and germ cells was based on previous description (Cheng et al. 1986) with a modification. Briefly, decapsulated testes from d35 mouse were incubated with collagenase (0.5 mg/ml) for 15 min with gentle oscillation, then were filtered through 80-µm copper meshes to eliminate Leydig cells. Tubules were suspended in collagenase for another 20-min incubation to remove myoid cells. Tubules were then incubated with hyaluronidase (1 mg/ml) for 25 min with gentle oscillation and pipetting. The resulting cells were washed four times with F12/DMEM, then were plated on Lab-Tek Chamber slides (Nunc Inc.; Naperville, IL) at 4 x 106 cells/ml of F12/DMEM supplemented with sodium bicarbonate (1.2 mg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% FBS. Cells were maintained in a humidified atmosphere of 95% air: 5% CO2 (v/v) at 32C for 48 hr. Thereafter, Spermatogenic cells were collected by gentle pipetting from Sertoli cells and dropped onto poly-L-lysin-coated slides for immunostaining. The germ cells adhered to Sertoli cells were lysed by a hypotonic treatment with 20 mM Tris, pH 7.4, for 2.5 min. Sertoli cells were cultured for an additional 24 hr for immunocytochemistry staining.
Immunocytochemistry Staining
Briefly, the slides were washed with PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4) three times and were fixed with cold methanol at 20C for 1 min. The endogenous peroxidase activity was blocked by treatment with 0.3% H2O2 in methanol for 15 min using 0.3% Triton X-100 in PBS for 15 min to increase cellular permeability. After blocking by preincubation with 10% normal goat serum in PBS at room temperature for 1 hr, the slides were then incubated with anti-Tyro 3 or anti-Axl antibody at a dilution of 1:100 at 4C in a humidified chamber overnight. The succeeding procedures are identical to the corresponding ones in immunohistochemistry staining.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The Tyro 3 immunostaining of Sertoli cells was strongly positive during the whole process of the postnatal development from d3 to d35. No positive staining was detected in spermatogenic cells, peritubular myoid cells, or interstitial cells. Distribution of Tyro 3 in Sertoli cells varied with the testicular development. At d3 and d7 (Figures 2A and 2B), the entire Sertoli cell except for the nucleus was strongly stained. At d14 (Figure 2C), immunostaining was concentrated at the adlumenal compartment of seminiferous tubules, only weakly positive or no positive staining was detected in the basal compartment. At d21 and d35 (Figures 2D and 2E), protein Tyro 3 localized uniformly on the processes of Sertoli cells surrounding spermatocytes and spermatids. Negative or very weak immunostaining was seen in the basal compartment including spermatogonia and preleptotene spermatocytes. No positive staining was seen in the perinuclear region of the Sertoli cells.
|
|
Unlike Tyro 3 and Axl, at d3, protein Mer immunostaining was prominently observed in the cytoplasm of primitive spermatogonia and of some interstitial cells and was only weakly detectable in Sertoli cells (Figure 5A). Based on morphology, immunoreactive positive interstitial cells were Leydig cells. In d7 testis, the Mer expression pattern was identical (not shown). However, from d14, spermatogonia became negative, whereas a strong cytoplasmic staining persisted through d35 in the Leydig cells (Figures 5B and 5C). A relatively weak immunostaining was also observed in the processes of Sertoli cells between spermatocytes throughout postnatal testis development.
|
To make comparative evaluation of the expression level in different testicular cells, the testes from 7-day-old mice were used for a semiquantitative evaluation of immunoexpression of Tyro 3, Axl, Mer, and Gas6. The testes from three animals and five sections from each testis were examined for each protein. The relative intensity of the immunostaining (brown staining) was evaluated as negative (), weak positive (+), or strong positive (+++). The similar immunostaining phenotype in all sections of different animals for each protein appeared. The results are shown in Table 1.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results demonstrated that Tyro 3 and Axl were expressed in Sertoli cells and their expression intensities did not show obvious changes during postnatal testicular development. We have also shown that the expression of Tyro 3 and Axl showed a stage-dependent pattern in mature testis. Expression of Mer was mainly observed in Leydig cells and primitive spermatogonia, whereas intense immunostaining of Gas6 was only evident in Leydig cells. In contrast with Tyro 3 and Axl, both Mer and Gas6 showed relative weak expression in Sertoli cells. This is the first time the details of the immunoexpression patterns of Tyro 3 subfamily receptors and their ligand Gas6 during development of mouse testis after birth have been examined.
Although a few previous studies have examined the expression of Tyro 3, Axl, Mer, and Gas6 mRNA by RT-PCR and Northern blot (Chan et al. 2000; Wong and Lee 2002
), the results were not consistent. Wong and Lee (2002)
reported that the expression of Tyro 3 and Axl decreased during postnatal testicular development, and Chan et al. (2000)
concluded that Axl was not expressed in Sertoli cells. However, it must be emphasized that Wong's study was performed by using total RNA extracted from testes of different age mice. In neonatal testis, the seminiferous tubules are mainly composed of Sertoli cells; the percentage of Sertoli cells in developing and mature testes are obviously decreased because of the increasing number of spermatogenic cells. Therefore, Wong's results probably reflect the reduced ratio of Sertoli cells versus germ cells during testicular development, instead of the actual expression levels of Tyro 3 and Axl in Sertoli cells. As for Chan et al.'s results, they were acquired from testicular clonal cell lines in which the expression of Tyro 3, Axl, and Mer might not really reflect the expression in vivo. Our study represents the first analysis of Tyro 3, Axl, and Mer proteins by immunostaining of developing testes, whereas previous analyses were performed at the mRNA level.
Although Tyro 3, Axl, and Mer belong to the same family and share high-sequence homology, they have distinct localization in testicular cells. Tyro 3 was expressed only in Sertoli cells, Axl was expressed in Sertoli cells and some Leydig cells, and Mer was expressed mainly in Leydig cells and in primitive spermatogonia. Sertoli cells and Leydig cells are the main somatic cells in the testis and play important roles in testicular development and spermatogenesis. As a nurturer, Sertoli cells provide essentially physical and trophic support for developing spermatogenic cells. Sertoli cells can also secrete an androgen-binding protein, which is necessary for spermatogenesis, under the control of follicle-stimulating hormone and testosterone. Leydig cells in interstitial tissue of testis can produce the male hormone testosterone under the control of luteinizing hormone. It has been reported (Lu et al. 1999) that Tyro 3, Axl, and Mer genes must all be disrupted for maximal degeneration of spermatogenesis. Considering our present results, we postulate that Tyro 3, Axl, and Mer may each play different roles and collaboratively control spermatogenesis.
Mammalian spermatogenesis is a highly synchronized, regular, long, and extremely complex process of cellular differentiation by which a spermatogonial "stem cell" is gradually transformed into highly differentiated haploid spermatozoa. In adult mammals, spermatogenesis is a continuous process that can be divided into three distinct phases (mitosis, meiosis, and spermiogenesis), each characterized by specific morphological and biochemical changes of nuclear and cytoplasmic components. Interestingly, our results showed that distribution of Tyro 3 or Axl in Sertoli cells varied with the testicular development and spermatogenesis. At postnatal d3 and d7, when Sertoli cells and spermatogonia proliferate, Tyro 3 and Axl were distributed uniformly in the plasma membrane and cytoplasm of Sertoli cells, suggesting that they may play roles in regulation the proliferation of spermatogonia and Sertoli cells in early stage of testis. At postnatal d14, when mature Sertoli cell function began to develop (e.g., the formation of the bloodtestis barrier, the expression of androgen-binding protein, the production of seminiferous fluid) (Gondos and Berndtson 1993), Tyro 3 and Axl mainly concentrated on the adlumenal compartment of seminiferous tubules. At postnatal d21, with the first wave of spermatogenesis, Tyro 3 and Axl were localized in the processes of Sertoli cells that surrounded the spermatocytes and round spermatids. At d35 and after, the expression of Tyro 3 and Axl became stage-dependent in Sertoli cells. Immunostaining was stronger in stages IVIII when many round spermatids and elongating spermatids are developing. At stages VII and VIII, positive staining mainly focused on the regions near the lumen of seminiferous tubules where elongating spermatids contacted the processes of Sertoli cells by apical ectoplasmic specializations. This periodic cycling with tubule stage is a property exhibited by many other Sertoli cell gene products, including the transcription factor GATA-1, which is expressed only at stages VIIVIII (Yomogida et al. 1994
) and androgen receptor, which has a highest expression at stages VIIVIII (Bremner et al. 1994
). The stage-dependent expression patterns of Tyro 3 and Axl suggest that they may involved in the differentiation of Sertoli cells to regulate spermatogenesis.
A few previous studies have shown Mer-mediated phagocytosis and clearance of apoptotic cells by macrophage (Scott et al. 2001; Cohen et al. 2002
). In testis, Sertoli cells are special cells and function as macrophage by maintaining the integrity of the seminiferous epithelium. During spermatogenesis, Sertoli cells phagocytose apoptotic germ cells and residual bodies; therefore, it is worthwhile to investigate whether the receptors play roles in regulating the phagocytosis of Sertoli cells.
As the common ligand for Tyro 3, Axl, and Mer, the immunoexpression of Gas6 in testis was also examined. The expression pattern of Gas6 in mouse testis was previously reported by Lu et al. (1999). In agreement with their results, we observed that Gas6 is mainly expressed in Leydig cells. However, we did not observe the periodic cycling of Gas6 in Sertoli cells as they observed by in situ hybridization. Actually, only a relatively weak immunostaining signal was detected in Sertoli cells in our present study. The controversy with the results of Lu et al. may depend on the fact that we studied the protein expression by immunohistochemistry, whereas they analyzed mRNA by in situ hybridization. It has been reported that Gas6 was a potential growth factor that stimulated human Schwann cell proliferation through the Axl/Tyro 3 tyrosine kinase receptors (Li et al. 1996
). Gas6 expression by Sertoli cells in vitro was stimulated by forskolin, and this forskolin-stimulated Gas6 expression was accompanied by an increase in Tyro 3 phosphorylation (Chan et al. 2000
). These previous studies suggest that Gas6 may exert its biological effects through autocrine or paracrine patterns. Gas6 expression pattern in testis suggests that it may play roles in spermatogenesis by regulating behaviors and functions of Sertoli cells.
Based on the structure motif, Tyro 3 family receptors have two potential functions: cell proliferation and adhesion. Previous studies have showed that Tyro 3 family is related to cell proliferation. In some malignant tumors, such as leukemia (O'Bryan et al. 1991), gastric cancer (Wu et al. 2002
), uterine leiomyoma (Sun et al. 2003a
), uterine endometrial cancer (Sun et al. 2003b
), and ovarian cancer (Sun et al. 2004
), the members of tyro3 receptor family and their ligand Gas6 were overexpressed. That Mer and Gas6 express in spermatogonia suggests that they may relate to proliferation of spermatogonia. A few studies also confirmed that the Tyro 3 family could induce homophilic binding independent of ligand in fibroblasts (Bellosta et al. 1995
) or mediated by soluble Gas6 in the 32D myeloid cell line (McCloskey et al. 1997
). Adherens junctions between Sertoli cells and spermatogenic cells are crucial not only for mechanical adhesion, but also for cell morphogenesis and differentiation during spermatogenesis. In the testis of triple mutant (Axl/Tyro3/Mer/) mice that completely lose spermiogenesis, no obvious changes in amounts of Sertoli cells in which Axl and Tyro 3 were expressed and of Leydig cells in which Mer was expressed were observed, whereas apoptotic spermatogenic cells apparently increased (Lu et al. 1999
), suggesting that proliferation of Sertoli cells and Leydig cells was not affected apparently by Tyro 3 family. The present study shows that Axl and Rse are mainly distributed in the processes of Sertoli cells that contact spermatogenic cells, particularly with round and elongating spermatids. Based on these observations, it would be interesting to ask if the Tyro 3 family receptors are involved in adhesion between Sertoli cells and germ cells. We are investigating this possibility.
In summary, in the present study, we have clearly shown that Tyro 3 family receptors and their ligand Gas6 are differentially expressed at the protein level in different testicular cells. Based on previous studies, these observations further suggest that Tyro 3 family receptors and their ligand Gas6 may participate in testicular development and spermatogenesis by regulating the function of Sertoli cells and Leydig cells. Our results provide clues to further study the mechanism of Tyro 3 family in regulating spermatogenesis.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ariyaratne HB, Mendis-Handagama SM, Mason JI (2000) Effects of tri-iodothyronine on testicular interstitial cells and androgen secretory capacity of the prepubertal Rat. Biol Reprod 63:493502
Bellosta P, Costa M, Lin DA, Basilico C (1995) The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding. Mol Cell Biol 5:614625
Bremner WJ, Millar MR, Sharpe RM, Saunders PT (1994) Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 135:12271234[Abstract]
Browne ES, Sohal GS, Bhalla VK (1990) Characterization of functional Leydig cells after purification on a continuous Percoll gradient. J Androl 11:379389
Chan MC, Mather JP, McCray G, Lee WM (2000) Identification and regulation of receptor tyrosine kinases Rse and Mer and their ligand Gas6 in testicular somatic cells. J Androl 21:291302
Chen J, Carey K, Godowski PJ (1997) Identification of Gas6 as a ligand for Mer, a neural cell adhesion molecule related receptor tyrosine kinase implicated in cellular transformation. Oncogene 14:20332039[CrossRef][Medline]
Cheng CY, Mather JP, Byer AL, Bardin CW (1986) Identification of hormonally responsive proteins in primary Sertoli cell culture medium by anion-exchange high performance liquid chromatography. Endocrinology 118:480488[Abstract]
Cohen PL, Caricchio R, Abraham V, Camenisch TD, Jennette JC, Roubey RA, Earp HS, et al. (2002) Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase. J Exp Med 196:135140
Crosier KE, Hall LR, Vitas MR, Crosier PS (1997) Expression and functional analysis of two isoforms of the human GM-CSF receptor alpha chain in myeloid development and leukaemia. Br J Haematol 98:540548[CrossRef][Medline]
Dai W, Pan H, Hassanain H, Gupta SL, Murphy MJ (1994) Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis. Oncogene 9:975979[Medline]
Fujimoto J, Yamamoto T (1994) Brt, a mouse gene encoding a novel receptor-type protein-tyrosine kinase, is preferentially expressed in the brain. Oncogene 9:693698[Medline]
Godowski PJ, Mark MR, Chen J, Sadick MD, Raab H, Hammonds RG (1995) Reevaluation of the roles of protein S and Gas6 as ligands for the receptor tyrosine kinase Rse/Tyro 3. Cell 82:355358[CrossRef][Medline]
Gondos B, Berndtson WE (1993) Postnatal and pubertal development. In Russell LD and Griswold MD, eds. The Sertoli Cell. Clearwater, FL, Cache River Press, 115154
Goruppi S, Ruaro E, Schneider C (1996) Gas6, the ligand of Axl tyrosine kinase receptor, has mitogenic and survival activities for serum starved NIH3T3 fibroblasts. Oncogene 12:471480[Medline]
Graham DK, Dawson TL, Mullaney DL, Snodgrass HR, Earp HS (1994) Cloning and mRNA expression analysis of a novel human protooncogene, c-mer. Cell Growth Differ 5:647657
Graham DK, Bowman GW, Dawson TL, Stanford WL, Earp HS, Snodgrass HR (1995) Cloning and developmental expression analysis of the murine c-mer tyrosine kinase. Oncogene 10:23492359[Medline]
Janssen JW, Schulz AS, Steenvoorden AC, Schmidberger M, Strehl S, Ambros PF, Bartram CR (1991) A novel putative tyrosine kinase receptor with oncogenic potential. Oncogene 6:21132120[Medline]
Jia R, Hanafusa H (1994) The proto-oncogene of v-eyk (v-ryk) is a novel receptor-type protein tyrosine kinase with extracellular Ig/GN-III domains. J Biol Chem 269:18391844
Konishi A, Aizawa T, Mohan A, Korshunov VA, Berk BC (2004) Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells. J Biol Chem 279:2876628770
Lai C, Lemke G (1991) An extended family of protein-tyrosine kinase genes differentially expressed in the vertebrate nervous system. Neuron 6:691704[CrossRef][Medline]
Lai C, Gore M, Lemke G (1994) Structure, expression, and activity of Tyro 3, a neural adhesion-related receptor tyrosine kinase. Oncogene 9:25672578[Medline]
Li R, Chen J, Hammonds G, Phillips H, Armanini M, Wood P, Bunge R, et al. (1996) Identification of Gas6 as a growth factor for human Schwann cells. J Neurosci 16:20122019[Abstract]
Lu Q, Gore M, Zhang Q, Camenisch T, Boast S, Casagranda F, Lai C, et al. (1999) Tyro-3 family receptors are essential regulators of mammalian spermatogenesis. Nature 398:723728[CrossRef][Medline]
Lu Q, Lemke G (2001) Homeostatic regulation of the immune system by receptor tyrosine kinases of the Tyro 3 family. Science 293:306311
Manfioletti G, Brancolini C, Avanzi G, Schneider C (1993) The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol 13:49764985[Abstract]
Mark MR, Scadden DT, Wang Z, Gu Q, Goddard A, Godowski PJ (1994) Rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain. J Biol Chem 269:1072010728
Mark MR, Chen J, Hammonds RG, Sadick M, Godowsk PJ (1996) Characterization of Gas6, a member of the superfamily of G domain-containing proteins, as a ligand for Rse and Axl. J Biol Chem 271:97859789
McCloskey P, Fridell YW, Attar E, Villa J, Jin Y, Varnum B, Liu ET (1997) GAS6 mediates adhesion of cells expressing the receptor tyrosine kinase Axl. J Biol Chem 272:2328523291
O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, Espinosa R 3rd, et al. (1991) Axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 11:50165031[Medline]
Ohashi K, Mizuno K, Kuma K, Miyata T, Nakamura T (1994) Cloning of the cDNA for a novel receptor tyrosine kinase, Sky, predominantly expressed in brain. Oncogene 9:699705[Medline]
Prieto AL, Weber JL, Lai C (2000) Expression of the receptor protein-tyrosine kinases Tyro-3, Axl, and mer in the developing rat central nervous system. J Comp Neurol 425:295314[CrossRef][Medline]
Rao Z, Handford P, Mayhew M, Knott V, Brownlee GG, Stuart D (1995) The structure of a Ca (2+)-binding epidermal growth factor-like domain: its role in protein-protein interactions. Cell 82:131141[CrossRef][Medline]
Rescigno J, Mansukhani A, Basilico C (1991) A putative receptor tyrosine kinase with unique structural topology. Oncogene 6:19091913[Medline]
Schulz NT, Paulhiac CI, Lee L, Zhou R (1995) Isolation and expression analysis of tyro3, a murine growth factor receptor tyrosine kinase preferentially expressed in adult brain. Brain Res Mol Brain Res 28:273280[CrossRef][Medline]
Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, Earp HS, et al. (2001) Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411:207211[CrossRef][Medline]
Sipahutar H, Sourdaine P, Moslemi S, Plainfosse B, Seralini GE (2003) Immunolocalization of aromatase in stallion Leydig cells and seminiferous tubules. J Histochem Cytochem 51:311318
Sugo T, Dahlback B, Holmgren A, Stenflo J (1986) Calcium binding of bovine protein S. Effect of thrombin cleavage and removal of the gamma-carboxyglutamic acid-containing region. J Biol Chem 261:51165120
Sun WS, Fujimoto J, Tamaya T (2003a) Coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine endometrial cancers. Ann Oncol 14:898906
Sun WS, Fujimoto J, Tamaya T (2003b) Clinical implications of coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine leiomyoma. Mol Hum Reprod 9:701707
Sun WS, Fujimoto J, Tamaya T (2004) Coexpression of Gas6/Axl in human ovarian cancers. Oncology 66:450457[CrossRef][Medline]
Taylor IC, Roy S, Yaswen P, Stampfer MR, Varmus HE (1995) Mouse mammary tumors express elevated levels of RNA encoding the murine homology of SKY, a putative receptor tyrosine kinase. J Biol Chem 270:68726880
Valverde P, Obin MS, Taylor A (2004) Role of Gas6/Axl signaling in lens epithelial cell proliferation and survival. Exp Eye Res 78:2737[CrossRef][Medline]
Varnum BC, Young C, Elliott G, Garcia A, Bartley TD, Fridell YW, Hunt RW, et al. (1995) Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6. Nature 373:623626[CrossRef][Medline]
Wong CC, Lee WM (2002) The proximal cis-acting elements Sp1, Sp3 and E2F regulate mouse mer gene transcription in Sertoli cells. Eur J Biochem 269:37893800
Wu CW, Li AF, Chi CW, Lai CH, Huang CL, Lo SS, Lui WY, et al. (2002) Clinical significance of AXL kinase family in gastric cancer. Anticancer Res 22:10711078[Medline]
Yomogida K, Ohtani H, Harigae H, Ito E, Nishimune Y, Engel JD, Yamamoto M (1994) Developmental stage- and spermatogenic cycle-specific expression of transcription factor GATA-1 in mouse Sertoli cells. Development 120:17591766
|