Structural determinants of heparan sulphate modulation of GDNF signalling

Glial cell line-derived neurotrophic factor (GDNF) has many functions including regulation of kidney morphogenesis and of neuron growth and survival in the enteric, sensory and central nervous systems. Reports of GDNF being used against Parkinson's disease in human patients have sparked intense clinical interest in GDNF signalling. We recently showed that GDNF signalling requires cell surface heparan sulphate glycosaminoglycans (Barnett et al., 2002, J. Cell Sci. 115, 4495-4503). Here we use exogenous modified heparins to determine those structural features required to inhibit GDNF signalling in ex vivo assays. 2-O-sulphate groups were found to impart high activity but were not absolute requirements for the inhibition of GDNF signalling. These findings may explain the similarities between the phenotypes of transgenic mice lacking GDNF and those lacking heparan sulphate 2-sulphotransferase, the enzyme responsible for achieving 2-O-sulphation of uronic acids in vivo.     

Glial‐derived neurotrophic factor regulates intestinal epithelial barrier function and inflammation and is therapeutic for murine colitis

Although enteric glial cells (EGCs) have been demonstrated to play a key role in maintaining intestinal epithelial barrier integrity, it is not known how EGCs regulate this integrity. We therefore hypothesized that glial‐derived neurotrophic factor (GDNF) produced by EGCs might be involved in this regulation. Here we investigated the role of GDNF in regulating epithelial barrier function in vivo. Recombinant adenoviral vectors encoding GDNF (Ad‐GDNF) were administered intracolonically in experimental colitis induced by dextran sulphate sodium (DSS). The disease activity index (DAI) and histological score were measured. Epithelial permeability was assayed using Evans blue dye. The anti‐apoptotic potency of GDNF in vivo was evaluated. The expression of tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and myeloperoxidase (MPO) activity were measured by ELISA assay and/or RT‐PCR. The expression of ZO‐1, Akt, caspase‐3, and NF‐κB p65 was analysed by western blot assay. Our results showed that GDNF resulted in a significant reduction in enhanced permeability, inhibited MPO activity, IL‐1β and TNF‐α expression, and increased ZO‐1 and Akt expression. Moreover, GDNF strongly prevented apoptosis in vivo and significantly ameliorated experimental colitis. Our findings indicate that GDNF participates directly in restoring epithelial barrier function in vivo via reduction of increased epithelial permeability and inhibition of mucosal inflammatory response, and is efficacious in DSS‐induced colitis. These findings support the notion that EGCs are able to regulate intestinal epithelial barrier integrity indirectly via their release of GDNF in vivo. GDNF is namely an important mediator of the cross‐talk between EGCs and mucosal epithelial cells. GDNF may be a useful therapeutic approach to the treatment of inflammatory bowel disease. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Glial cell line-derived neurotrophic factor in Purkinje cells of adult zebrafish: An autocrine mode of action?

Glial cell line-derived neurotrophic factor (GDNF) has a neuroprotective role in Purkinje cells of cerebellum, promoting the survival and the differentiation of these cells. Its signalling is mediated by a receptorial complex GFRalpha1/RET. In the brain of adult zebrafish (Danio rerio) we previously investigated GDNF expression and localization, but no data exist regarding GFRalpha1 and RET presence. Thus, the present study was designed to clarify the morphological relation between GDNF and its receptorial complex GFRalpha1/RET immunoreactivity in the cerebellum of adult zebrafish. The expression of gdnf, GFRalpha1 and ret genes was demonstrated in adult zebrafish cerebellum by a standard RT-PCR. The distribution of GDNF and its receptorial complex GFRalpha1/RET was examined by single and double immunocytochemical stainings. In the valvula and corpus cerebelli GDNF, GFRalpha1 and RET immunoreactivity was seen co-localized in Purkinje cells, identified morphologically and by using an antiserum against a specific marker for these cells, aldolase C enzyme. In the vestibulolateralis lobe, Purkinje neurons were lacking in both the eminentiae granulares and medial caudal lobe. These results demonstrated the expression of the GDNF receptorial complex in adult zebrafish cerebellum and suggest an autocrine mode of action of GDNF in Purkinje cells.     

COMMENTS

The molecular basis of testicular germ cell tumourigenesis are not well elucidated. Growth factors regulate cell growth, differentiation and apoptosis. Major families of growth factors are present in the male gonad from early fetal development to adult life. They are involved in germ cell proliferation and differentiation. Growth signalling pathways suffer deregulation in many human malignancies. Given the importance of growth signals in normal testicular development and their acquired deregulation in most human cancers, growth factors and signalling molecules that have been implicated in the genesis of testicular germ cell tumours, are reviewed. We detected a somatic mutation of SMAD4 gene, responsible for loss of protein function in seminomas. This mutational inactivation may affect the activity of several members of TGFbeta superfamily (TGFbeta, activin, inhibin, BMP). VEGF expression has been shown to predict metastasis in seminomas. A significant association of HST-1 expression, a member of fibroblast growth factors, with the nonseminomatous phenotype and with tumour stage has been described. In contrast, C-KIT is expressed by seminomas only, from the preinvasive stage. Despite intense expression in almost all seminomas, activating mutation of C-KIT gene is seldom reported. Recently, the first animal model of classical testicular seminoma has been identified in transgenic mouse overexpressing GDNF. RET (GDNF receptor) expression is demonstrated in human seminomas, and not in nonseminomatous tumours. However, the exact molecular alterations of GDNF/RET/GFRalpha1 complex in germ cell tumours are not known. Finally, beside growth factors, other signalling molecules such as peptide hormones may be involved in testicular carcinogenesis. We have demonstrated a specific pattern of somatostatin receptors expression in each type of testicular germ cell tumours, with a loss of sst3 and sst4 in seminomas and loss of sst4 and expression of sst1 in nonseminomas only. These data suggest an antiproliferative action of somatostatin in testicular cancers. In summary, many growth factors and signalling molecules seem to represent specific markers for different histological types of germ cell tumours (seminomas versus nonseminomas) and may play a role in the differentiation of germ cell tumours. Despite a complex signalling pathway involved in the physiological functions of male gonad, little is known about the implication of this signalling network in testicular malignancies. From a practical stand-point, further studies on the role of growth factors in human germ cell tumours may offer a new therapeutical perspective with the development of specific pharmacological signalling modulators that could be used as therapeutic agents.     

Comparative study on Lewis lung tumor lines with ‘low’ and ‘high’ metastatic capacity. II. Cytochemical and biochemical evidence for differences in glycosaminoglycans

The enhanced metastatic capacity of an in vivo selected Lewis lung tumor line (LLT-HH) was correlated with changes in cell-associated glycosaminoglycans (GAG) using ultrastructural cytochemistry, flow cytometry and biochemistry. The increase in highly sulphated GAG content on the cell membrane of LLT-HH cells compared to the parent LLT cells was demonstrated cytochemically. Using in vitro [³H]glucosamine labelling of GAG components it was shown that the LLT-HH cells were characterized by a high production of heparan sulphate while the parent LLT line had a high hyaluronic acid-chondroitin sulphate production. The high metastatic phenotype is accompanied by an altered production of cell-associated GAGs.     

Association study between plasma GDNF and cognitive function in late-onset depression

Objective

Improving evidence suggest that neurotrophic growth factor systems might be involved in the pathophysiology of major depressive disorder (MDD). The glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor from the transforming growth factor-β-family, which plays a role in the development and function of hippocampal cells. This study was aimed to test whether GDNF in plasma was abnormal in late-onset depression (LOD), and whether it was associated with the cognitive impairment of LOD.

Methods

The plasma GDNF levels in LOD patients (n = 27) before antidepressant treatment and normal control subjects (n = 28) were measured with the ELISA method. All subjects were assessed by neuropsychological tests and Hamilton Depression Rating Scale (HDRS).

Results

The performance of neuropsychological tests of the LOD group except TMT-B was significantly poorer than those of the control group. The plasma GDNF levels in LOD patients were significantly increased compared to control subjects (P < 0.05). Furthermore, the increase of plasma GDNF level was significantly positively correlated with Digit Span Test backward score in LOD patients, and negatively associated with TMT-B performance.

Conclusions

The findings suggest that LOD patients in acute phase have extensive impairments of cognitive function, and higher plasma GDNF might be involved in the pathogenesis of LOD, which may be associated with the cognitive dysfunction in LOD.     

The reduction in immunogenicity of neurotrophin overexpressing stem cells after intra-striatal transplantation by encapsulation in an in situ gelling collagen hydrogel

Delivery of neurotrophic factors to the brain via genetically modified bone marrow-derived mesenchymal stem cells (MSCs) offers a promising neuroprotective strategy for neurodegenerative diseases. However, MSCs delivered to the CNS typically show poor survival post-transplantation, which is accompanied by microglial activation and astrocyte recruitment at the graft site. Recent studies have shown the potential of biomaterials to provide a supportive matrix for transplanted cells which may assist in the grafting process. In this study, an in situ gelling type I collagen hydrogel was evaluated as an intracerebral transplantation matrix for delivery of glial cell line-derived neurotrophic factor (GDNF)-overexpressing MSCs to the rat brain (GDNF-MSCs). In vitro analyses demonstrated that this collagen hydrogel did not affect the viability of the GDNF-MSCs nor did it prevent GDNF secretion into the surrounding medium. In vivo analyses also confirmed that the collagen hydrogel did not negatively impact on the survival of the cells and permitted GDNF secretion into the striatal parenchyma. Importantly, this study also revealed that transplanting GDNF-MSCs in a collagen hydrogel significantly diminished the host brain's response to the cells by reducing the recruitment of both microglia and astrocytes at the site of delivery. In conclusion, this hydrogel, which is composed of the natural extracellular matrix, collagen, was shown to be a well-tolerated cell delivery platform technology which could be functionalised to further aid cell support and graft integration.     

Theret-activating ligand GDNF is differentiative and not mitogenic for normal and neoplastic human chromaffin cells in vitro

Activating mutations of the receptor tyrosine kinase,ret, are associated with multiple endocrine neoplasia type 2A (MEN-2A). However, the mechanisms leading to tumor development are unclear. Glial-derived neurotrophic factor (GDNF) activates wild-typeret via interaction with a second receptor, GFR α-1. We have utilized GDNF to stimulate normal and neoplastic chromaffin cells in order to ask whetherret activation is mitogenic. Cells from three normal adult adrenal medullas, one sporadic pheochromocytoma, and three MEN-2A pheochromocytomas were labeled with bromodeoxyuridine (BrdU) for 12 d in the presence or absence of GDNF or nerve growth factor (NGF), which is known to stimulate neurite outgrowth, but not proliferation in human chromaffin and pheochromocytoma cell cultures. Responses to GDNF and NGF were comparable, except for two MEN-2A pheochromocytomas that responded minimally to GDNF and robustly to NGF. These tumors responded to GDNF biochemically, as measured by phosphorylation of mitogen-activated protein kineses, despite their weak morphological responses. Our findings suggest that activation ofret may not be sufficient to produce chromaffin cell hyperplasia or neoplasia directly by stimulating cell proliferation. However, the possibility that altered cell-cell or cell-substrate interactions might cause responses to become differentiative rather than proliferative in vitro has not been ruled out. We also demonstrate, for the first time, that at least some human pheochromocytomas with an MEN-2Aret mutation respond to a normalret ligand. This responsiveness could be mediated by a remaining normalret allele or by other mechanisms.     

Enhanced dopamine transporter activity in middle-aged Gdnf heterozygous mice

Glial cell line-derived neurotrophic factor (GDNF) supports the viability of midbrain dopamine (DA) neurons that degenerate in Parkinson's disease. Middle-aged, 12 month old, Gdnf heterozygous (Gdnf^+/-) mice have diminished spontaneous locomotor activity and enhanced synaptosomal DA uptake compared with wild type mice. In this study, dopamine transporter (DAT) function in middle-aged, 12 month old Gdnf^+/- mice was more thoroughly investigated using in vivo electrochemistry. Gdnf^+/- mice injected with the DAT inhibitor, nomifensine, exhibited significantly more locomotor activity than wild type mice. In vivo electrochemistry with carbon fiber microelectrodes demonstrated enhanced clearance of DA in the striatum of Gdnf^+/- mice, suggesting greater surface expression of DAT than in wild type littermates. Additionally, 12 month old Gdnf^+/- mice expressed greater D₂ receptor mRNA and protein in the striatum than wild type mice. Neurochemical analyses of striatal tissue samples indicated significant reductions in DA and a faster DA metabolic rate in Gdnf^+/- mice than in wild type mice. Altogether, these data support an important role for GDNF in the regulation of uptake, synthesis, and metabolism of DA during aging.     

Laminin and heparan sulphate proteoglycan in the lesioned adult mammalian central nervous system and their possible relationship to axonal sprouting

Summary Severalin vitro studies indicate that the extracellular matrix (ECM) glycoprotein laminin can promote neurite outgrowth from CNS (central nervous system) neurons. Laminin has been detected immunohistochemically in astrocytes in the embryonic but not the uninjured adult mammalian CNS. In the injured adult CNS, it is found in some reactive astrocytes located near the site of CNS lesions. In the present study, we have attempted to examine the relationship between these laminin⁺ astrocytes and the axonal sprouting that occurs after CNS injuries. This was studied in the intracranially transected adult rat optic nerve which consists of a cranial segment devoid of all retinal ganglion cell axons, and a retinal segment attached to the retina which contains some viable axons that undergo sprouting. Laminin⁺ reactive astrocytes were found in the cranial segment, but not in the retinal segment. In addition, the cut ends of the retinal and cranial segments were capped by an intensely laminin⁺, glial fibrillary acidic protein negative (GFAP) region. Axonal sprouts from the transected retinal ganglion cell axons, identified by anterogradely transported rhodamine isothiocynate (RITC), were confined to laminin^–, GFAP⁺ regions of the retinal segment. These results suggest that injury-induced axonal sprouting in the adult mammalian CNSin vivo may be promoted by molecules other than laminin, that may be associated with astrocytes.The presence of heparan sulphate proteoglycan HSP G was also examined in the transected optic nerve because the neurite outgrowth promoting factors found in conditioned media derived from several cell typesin vitro have been shown to consist of a complex of laminin and heparan sulphate proteoglycan. No significant changes in heparan sulphate proteoglycan-like immunoreactivity was observed after transection. The presence of laminin and HSPG were also examined in the lesioned adult rat cerebral cortex.     

Time-course of GDNF and its receptor expression after brain injury in the rat

The aim of the present work was to perform, by in situ hybridization, a time-course analysis of the glial cell line-derived neurotrophic factor (GDNF) and its receptor mRNA expression in two models of brain injury in the rat: (a) excitotoxic lesion by ibotenic acid injection in the hippocampal formation; (b) mechanical lesion by needle insertion through the cerebral cortex including the white matter of the corpus callosum. The time-course analysis, ranging from 6 h to 8 days, showed that the GDNF and its receptor (RET, GFRα-1 and GFRα-2) mRNA expressions were differentially up-regulated in both models of lesion. This in vivo regulation of the GDNF and its receptor mRNA expression indicates their involvement in the process of neuronal protection and regeneration occurring after brain injury.     

A dual-hit animal model for age-related parkinsonism

Parkinson's disease is a neurological disorder which afflicts an increasing number of individuals. If the wider complex of extrapyramidal symptoms referred to as “age-related parkinsonism” is included, the incidence is near 50% of the population above 80 years of age. This review summarizes recent studies from our laboratories as well as other research groups in the quest to explore the multi-faceted etiology of age-related neurodegeneration, in general, and degeneration of the substantia nigra dopaminergic neurons, in particular. Our work during recent years has focused on assessment of potential interactive effects of a reduction in glial cell line-derived neurotrophic factor (GDNF) and the aging process (intrinsic factors) and early neurotoxin exposure (an extrinsic factor) on dopamine (DA) systems and the behaviors they mediate. The guiding hypothesis directing the research to be described was that a combination of the two factors would exacerbate the decline in the DA transmitter system function that occurs during aging. The results obtained were consistent with the well-established aging-related decline in function and structure of neurons utilizing DA as a transmitter and motor function, and extended knowledge by establishing that the genetic reduction of Gdnf exacerbated these aging related changes. Thus, GDNF reduction appears to increase the vulnerability of the DA neurons to the many different challenges associated with the aging process. Assessment of methamphetamine effects on young Gdnf^+/− mice indicated that reduced GDNF availability increased the vulnerability of DA systems to this well-established neurotoxin. The work discussed in this review is consistent with earlier work demonstrating the importance of GDNF for maintenance of DA neurons and also provides a novel model for progressive DA degeneration and motor dysfunction.     

THE EFFECT OF VARIOUS FORMS OF HEPARIN ON THE RELEASE OF IMMUNE COMPLEXES FROM THE SURFACE OF CULTURED MESANGIAL CELLS

Heparin is capable of enhancing the rate of release of antigen from nephritic rat kidneys. It also interferes with the binding of immune complexes by cultured glomerular mesangial cells. Postulating that these two effects might be related, we sought to determine what basic aspects of the molecular structure of heparin are responsible for the interference with binding in vitro . After cultured mesangial cells had bound radiolabelled synthetic immune complexes, heparin or a variety of structurally related molecules were added to the supernatant. De-N-sulphated heparin, heparan sulphate, low molecular weight heparin, and low molecular weight dextran sulphate had no effect on immune complex binding. High molecular weight dextran sulphate was able, like heparin, to dislodge immune complexes from mesangial cells, suggesting that high molecular weight and high sulphation are required. These results differ from previous findings in vivo , suggesting that the effect of heparin in vivo is not due to interaction at the mesangial cell surface. Alternative explanations for the effect of heparin in the intact animal include destabilization of the immune complex structure or, more probably, an effect at the boundary between the immune complex deposit and the basement membrane.     

Acidic oligosaccharide sugar chain, a marine-derived oligosaccharide, activates human glial cell line-derived neurotrophic factor signaling

Gial derived neurotrophic factor (GDNF) modulates neuronal cell differentiation during development and protects against neurodegeneration by preventing apoptosis at maturity. GDNF's role in tissue maintenance has generated interest in the therapeutic potential of GDNF in treating neurological disorders such as Parkinson's disease. Heparan sulfate has been shown to be essential for GDNF signaling and altering the levels of heparan sulfate promotes or inhibits GDNF functional activity. To search for other oligosaccharides capable of modulating GDNF activity as potential therapeutic molecules, we investigated the effect of acidic oligosaccharide sugar chain (AOSC) and its sulfated derivative on GDNF induced neurotrophic events by using Western-blotting, immunofluorescence cell staining, and immunoprecipitation techniques in PC12 cells expressing the GDNF receptors GFR alpha 1-Ret. AOSC significantly improved the neurite outgrowth and activated c-Ret phosphorylation in PC12-GFR alpha 1-Ret cells, but its sulfated derivative inhibited GDNF activity. Studies to understand the opposing biological effects of AOSC and its sulfated derivative on GDNF activity demonstrated that reduced GDNF binding to PC12-GFR alpha 1-Ret cell surface in the presence of the sulfated derivative likely suppressed GDNF activity as both AOSC and its sulfated derivatives had similar binding affinities to GDNF. This study illustrates the importance of oligosaccharide structure and charge on influencing GDNF activity and the potential use of oligosaccharides in modulating GDNF activity for therapeutic purposes.     

Neurotropic growth factors and glycosaminoglycan based matrices to induce dopaminergic tissue formation

Current cell replacement therapies in Parkinson's disease (PD) are limited by low survival of transplanted cell and lacking regeneration of neuronal circuitries. Therefore, bioartificial cell carriers and growth/differentiation factors are applied to improve the integration of transplants and maximize newly generated and/or residual dopaminergic function. In this work, biohybrid poly(ethylene glycol) (starPEG)-heparin hydrogels releasing fibroblast growth factor 2 (FGF-2) and glial-derived neurotrophic factor (GDNF) were used to trigger dopaminergic tissue formation by primary murine midbrain cells in vitro. Matrix-delivered FGF-2 enhanced cell viability while release of GDNF had a pro-neuronal/dopaminergic effect. Combined delivery of both factors from the glycosaminoglycan-based matrices resulted in a tremendous improvement in survival and maturation capacity of dopaminergic neurons as obvious from tyrosine hydroxylase expression and neurite outgrowth. The reported data demonstrate that glycosaminoglycan-based hydrogels can facilitate the administration of neurotrophic factors and are therefore instrumental in potential future treatments of PD.     

One Step Enzyme Linked Immunosorbent Assay for Direct Estimation of Serum Testosterone

Abstract

One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of testosterone in human serum is described. Testosterone-3-O-carboxymethyl-oxime-bovine serum albumin (testosterone-3-O-CMO-BSA), was used as immunogen and testosterone-3-O-carboxymethyl-oxime-adipic-acid dihydrazide-horseradish peroxidase (testosterone-3-O-CMO-ADH-HRP) was used as tracer. To the testosterone antibody coated microtiter wells, standard or serum samples (100 µL), along with testosterone-3-O-CMO-ADH-HRP conjugate (100 µL) were incubated for 1 h at 37°C. Bound enzyme activity was measured by using tetra methyl benzidine/hydrogen peroxide (TMB/H₂O₂) as a substrate. In this new strategy, charcoal stripped pooled human serum spiked with non-cross reactive C₁₈, C₁₉, C₂₁, and C₂₇ steroids, used for preparing the standards and blocking the sex hormone binding globulin (SHBG)/and other steroid binding globulins (SBG). The sensitivity of the assay was 0.015 ng/mL. The intra-assay and inter-assay coefficients of variation (CVs) were ranged from 7.8 to 11.8 and 4.8 to 10.4, respectively. The serum testosterone values, obtained by this method, were correlated well with those obtained by radioimmunoassay r = .98 (n = 100).     

Increased glial‐derived neurotrophic factor in the small intestine of rats infected with the tapeworm,Hymenolepis diminuta

The neurotrophin, glial‐derived neurotrophic factor (GDNF), is essential for the development of the enteric nervous system (ENS) in both the embryo and neonate and may be important for maintenance and plasticity of ENS. The tapeworm, Hymenolepis diminuta, altered the number of cells containing GNDF in the host’s jejunum and ileum. Numbers and locations of GDNF‐containing cells were determined by applying monoclonal anti‐GDNF antibody to intestinal segments collected from infected and uninfected age‐matched rats during the initial 34 days post‐infection (dpi). Most cells staining positive for GDNF were present in the lamina propria of the jejunum and ileum from both infected and uninfected rats. The co‐localization of staining by the antibodies, anti‐GDNF and anti‐ED2 (a nuclear specific antibody for resident macrophages) indicated that at least 74% of the cells staining for GDNF were macrophages. Mast cells did not stain with the anti‐GDNF antibody. The increased number of GDNF+ cells in the infected rat intestine suggests that this neurotrophin may play a role in the neural and mucosal responses to lumenal tapeworm infection.     

In vitro inhibition of liver forms of the rodent malaria parasite Plasmodium berghei by naphthylisoquinoline alkaloids – structure-activity relationships of dioncophyllines A and C and ancistrocladine

Naphthylisoquinoline alkaloids are derived from Dioncophyllaceae and Ancistrocladaceae species and comprise a new class of promising antimalarials with a demonstrated potential against asexual erythrocytic Plasmodium falciparum and P. berghei stages in vitro. We report herein the pronounced activity of pure naphthylisoquinoline alkaloids against exoerythrocytic malaria parasites. P. berghei-infected human hepatoma cells (Hep G2) were incubated with culture medium containing selected alkaloids at 10 μg/ml. The most active compounds, showing inhibitory activity of more than 40%, were dioncophylline A (compound 1), dioncophyllacine A (compound 6), and ancistrobarterine A (compound 12). For structure-activity investigations of dioncophyllines A (compound 1) and C (compound 3) and ancistrocladine (compound 7) a selection of their analogs from natural or synthetic sources was examined. Dioncophylleine A (compound 16), 5′-O-demethyl-8-O-methyl-7-epi-dioncophylline A (compound 17), N-formyl-8-O-methyl-dioncophylline C (compound 21), and N-formyl-8-O-benzoyldioncophylline C (compound 24) were found to display high levels of activity as well, although the former two compounds caused damage to the host-cell monolayers. As naphthylisoquinoline alkaloids are also highly active against blood forms of Plasmodium spp., they should be regarded as lead compounds for further development as drugs against erythrocytic and exoerythrocytic stages of Plasmodium spp. Received: 28 January 1997 / Accepted: 17 March 1997     

Induction of Anchorage-Independent Growth by Amphiregulin

Abstract

We have previously shown that the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AR) exhibits low potency as a result of its C-terminal truncation. This led us to investigate whether its inability to promote anchorage-independent growth (AIG) of normal cells arose because of its compromised interaction with EGFR. Wild type AR., was tested in AIG and mitogenesis assays using NRK-49F or NR6/HER fibroblasts. In contrast to NR6/HER cells, the response of NRK-49F fibroblasts to AR was much lower than expected. As the effect of AR was heparin-insensitive, contributions from heparan sulphate proteoglycan interactions could not explain the differing sensitivities of the cells. Comparison of the effects of AR on two additional cell lines indicated that low EGFR number correlated with AR insensitivity: this suggested that the low potency of AR precluded activation of sufficient receptors to elicit a response.Consistent with this proposal, a modified form of AR (AR_1-90(leu86)) with enhanced potency was able to induce AIG of NRK-49F fibroblasts. Thus, the ability of AR to promote AIG is determined both by ligand potency and the EGFR complement of cells.