Peroxynitrite mediates VEGF's angiogenic signal and function via a nitration-independent mechanism in endothelial cells.

The modulation of angiogenic signaling by reactive oxygen species (ROS) is an emerging area of interest in cellular and vascular biology research. We provide evidence here that peroxynitrite, the powerful oxidizing and nitrating free radical, is critically involved in transduction of the VEGF signal. We tested the hypothesis that VEGF induces peroxynitrite formation, which causes tyrosine phosphorylation and mediates endothelial cell migration and tube formation, by studies of vascular endothelial cells in vitro and in a model of hypoxia-induced neovascularization in vivo. The specific peroxynitrite decomposition catalyst FeTPPs blocked VEGF-induced phosphorylation of VEGFR2 and c-Src and inhibited endothelial cell migration and tube formation. Furthermore, exogenous peroxynitrite mimicked VEGF activity in causing phosphorylation of VEGFR2 and stimulating endothelial cell growth and tube formation in vitro and new blood vessel growth in vivo. The selective nitration inhibitor epicatechin enhanced VEGF's angiogenic function in activating VEGFR2, c-Src, and promoting endothelial cell growth, migration, and tube formation in vitro and retinal neovascularization in vivo. Decomposing peroxynitrite with FeTPPs or blocking oxidation using the thiol donor NAC blocked VEGF's angiogenic functions in vitro and in vivo. In conclusion, peroxynitrite is critically involved in transducing VEGF's angiogenic signal via nitration-independent and oxidation-mediated tyrosine phosphorylation.

TGF-beta increases retinal endothelial cell permeability by increasing MMP-9: possible role of glial cells in endothelial barrier function.

PURPOSE: To determine transforming growth factor (TGF) beta effects on matrix metalloproteinases (MMPs) as a potential cause of the blood-retinal barrier breakdown at the onset of angiogenesis. Previously, glial cells were shown to play a role in the angiogenesis process and to express the angiogenic regulating factor TGF-beta, which becomes active under hypoxia conditions. Here, the authors demonstrate that retinal endothelial cells express MMP-9 when treated with TGF-beta or cocultured with glial cells and that both TGF-beta and MMP-9 increase endothelial cell permeability. METHODS: Primary cultures of bovine retinal endothelial (BRE) cells grown on porous membranes were treated with TGF-beta or purified MMP-9, and permeability changes were assayed. The amount and distribution of the tight junction protein occludin also was analyzed by immunocytochemistry and Western blotting. Cell extracts or conditioned media from TGF-beta-treated BRE cells and from glial cell-BRE cocultures were analyzed for MMP-9 content by substrate gel electrophoresis (zymography) or Western blotting. RESULTS: Both TGF-beta and MMP-9 increased the permeability of BRE monolayers and reduced the levels of the junction protein occludin. The effect of MMP-9 on permeability was rapid, but the TGF-beta-induced permeability required longer incubation and was blocked by anti-TGF-beta and anti-MMP-9 antibodies as well as by TGF-beta latency-associated peptide. Zymography showed that MMP-9 activity, which was very low or absent in untreated BRE cultures, was dramatically increased by TGF-beta as well as by coculturing with either astrocytes or Müller glial cells. Anti-TGF-beta antibody blocked the TGF-beta effect, but not the coculture effect on MMP-9 production. CONCLUSIONS: These data indicate a direct correlation between TGF-beta-induced MMP-9 activity and increased endothelial cell permeability. Moreover, endothelial cell production of MMP-9 is regulated by glial cells through expression of TGF-beta or by direct cell-to-cell contact. During retinal disease, glial cell production of active TGF-beta may contribute to breakdown of the blood-retinal barrier by stimulating endothelial cell MMP-9 production.

Steroid 5alpha-reductase 2 gene melting polymorphisms in male subjects with azoospermia or oligospermia.

OBJECTIVE: This study was designed to test the hypothesis that mutations in the gene for type 2 steroid 5alpha-reductase (SRD5A2) may be the cause of a phenotype characterized primarily by oligospermia or azoospermia. STUDY DESIGN: Deoxyribonucleic acid from control subjects and subjects with oligospermia (n = 12) and azoospermia (n = 6) were evaluated for mutations in SRD5A2. Methods used for mutation analysis included polymerase chain reaction, Southern blotting, and denaturing gradient gel electrophoresis. RESULTS: Denaturing gradient gel electrophoresis of all 5 amplified exons resulted in similar migration patterns in samples from both control and study subjects. Genomic deoxyribonucleic acid subjected to denaturing gradient gel electrophoresis after restriction digest revealed melting polymorphisms. Direct sequencing of the gene in a single patient with a unique melting polymorphism yielded a normal sequence. CONCLUSIONS: Melting polymorphisms for SRD5A2 were detected in a group of patients with oligospermia or azoospermia. Sequence analysis did not demonstrate functional mutations in the coding sequence of this gene.

VEGF-induced permeability increase is mediated by caveolae.

PURPOSE: To determine the cellular route by which vascular endothelial cell growth factor (VEGF) increases the permeability of cultured retinal endothelial cells and to test whether nitric oxide (NO) production by NO synthase (NOS) is involved in signaling VEGF's permeability enhancing effects. METHODS: Cultured bovine retinal microvascular endothelial (BRE) cells were used for bioassay of permeability function and its ultrastructural correlates. The role of NOS activity in VEGF's permeability enhancing effects was tested with the use of an NOS inhibitor. Because activity of endothelial NOS (eNOS) is thought to be regulated by its interaction with the caveolar protein caveolin-1, structural relationships between eNOS, caveolin-1, and the VEGF receptor FIk-1/KDR were analyzed with double-label immunofluorescence and cell fractionation procedures. RESULTS: Bioassays of permeability function and structure demonstrated that VEGF increases permeability of cultured BRE cells by an NOS-dependent process of transcytotic transport in caveolae. Double-label analysis showed that Flk-1/KDR and eNOS colocalize with caveolin-1 in plasma membrane caveolae. Cell fractionation and immunoblot analysis confirmed this effect. Densitometry showed that Flk-1/KDR, eNOS, and caveolin-1 levels were highest in caveolar fractions. Similar results were obtained in studies with bovine aortic endothelial cells. CONCLUSIONS: These results demonstrate that VEGF increases endothelial cell permeability by an eNOS-dependent mechanism of transcytosis in caveolae. Localization of Flk-1/KDR and eNOS with caveolin-1 suggests that VEGF signaling occurs within the caveolar compartment.

Effects of hypoxia on glial cell expression of angiogenesis-regulating factors VEGF and TGF-beta.

Perivascular glial cells are thought to be involved in physiologic vascularization and also in pathologic angiogenesis in the central nervous system. We have previously shown that astrocytes are a source of transforming growth factor-beta (TGF-beta) and another inhibiting factor, which block endothelial cell growth and induce their apoptosis. Astroglia are also known to express vascular endothelial growth factor (VEGF), which is up-regulated during hypoxia. Here we demonstrate the effects of hypoxia on the expression of both TGF-beta and VEGF by retinal glial cells. Muller cells isolated from rat retina were incubated under hypoxia or normoxia and the resulting conditioned media (H-MCM and N-MCM) were assayed for their effects on growth of bovine retinal capillary endothelial (BRE) and the TGF-beta-sensitive mink lung epithelial CCL cells. The expression and quantities of VEGF and TGF-beta (active vs. latent form) were determined by immuno-adsorption, Western or Northern blotting, and ELISA. N-MCM stimulated BRE cell growth by twofold but inhibited CCL cells under similar assay conditions, whereas H-MCM had a weak stimulating effect on BRE and substantial inhibitory activity on CCL cells. Adsorption of MCM by specific antibodies as well as Western and Northern blot analysis indicated that stimulating and inhibitory activities of MCM are due to the presence of VEGF and TGF-beta, respectively. ELISA revealed that the hypoxia condition converts latent TGF-beta into its active form. In N-MCM, TGF-beta is found predominantly in the latent form, but in hypoxia MCM it is mainly active. Furthermore, it was found that treatment of Muller cells with exogenous TGF-beta under either hypoxia or normoxia increases VEGF expression in a time- and dose-dependent fashion. TGF-beta activation may, therefore, be prerequisite for hypoxia-induced up-regulation of VEGF and stimulation of angiogenesis in vivo.

Angiostatic role of astrocytes: suppression of vascular endothelial cell growth by TGF-beta and other inhibitory factor(s).

Our previous in vivo analyses have suggested that astrocytes play a key role in retinal vascularization by inducing endothelial cell differentiation. Here we demonstrate that medium conditioned by cultured rat brain astrocytes (ACM) contains factors, including transforming growth factor-beta (TGF-beta), that inhibit endothelial cell growth. Serum-free medium conditioned for 1-3 days was tested on exponentially growing bovine retinal microvascular endothelial, aortic endothelial, mink lung epithelial CCL-64, and Swiss mouse 3T3 fibroblast cells. The growth of all four cell types was inhibited in a dose- and time-dependent manner. CCL cells, which are used as a model for assaying TGF-beta activity, were more sensitive than the endothelial cells, suggesting that ACM contains TGF-beta. Moreover, acid treatment significantly increased the inhibitory activity of ACM, indicating that TGF-beta in ACM is predominantly in the latent form. Mouse fibroblasts, which are not affected by TGF-beta treatment under the same conditions, were also inhibited by ACM. This suggests that other inhibitory factors in addition to TGF-beta may be involved. Adsorption by an anti-TGF-beta polyclonal antibody column substantially reduced but did not eliminate the inhibitory activity of ACM for CCL and endothelial cells. Western blot analysis of ACM and proteins eluted from the affinity column revealed a 25 kDa band that co-migrates with TGF-beta. Comparative densitometry of the 25 kDa bands on Western blot indicated that the amount of TGF-beta in ACM is not sufficient to account for the total growth-inhibitory activity. These experiments demonstrate directly that rat brain astrocytes express TGF-beta. They also indicate that astrocytes may produce other growth-inhibitory factor(s) yet to be identified.

The first analysis of exon 1 (the transactivation domain) of the androgen receptor gene in infertile men with oligospermia or azoospermia.

OBJECTIVE: To examine the role of the androgen receptor (AR) gene in spermatogenesis by evaluating infertile men with idiopathic oligospermia or azoospermia, with special emphasis on the transactivation domain (exon 1) of AR gene because it has not been studied in this population previously. STUDY DESIGN: A molecular study of the AR gene. Deoxyribonucleic acid samples were screened for possible AR gene mutations using polymerase chain reactions (PCR). SETTING: The offices and laboratories of the Medical College of Georgia. PARTICIPANTS: Infertile men with oligospermia or azoospermia and an otherwise negative laboratory evaluation. Controls consisted of healthy fertile men. MAIN OUTCOME MEASURES: Each exon (2 to 8) and each of five overlapping exon segments for exon 1 of the AR gene was amplified using PCR for each participant's DNA sample. The PCR products were evaluated by size using electrophoresis and a DNA size marker. RESULTS: Sixteen idiopathic oligospermic or azoospermic men entered the study. All seven exons and the five overlapping segments of exon 1 were amplified and were of the appropriate size on electrophoresis when compared with controls, the DNA size marker, and the exon sequence. CONCLUSIONS: Preliminary protein studies on AR suggested that up to 40% of infertile men may have AR abnormalities. Since the availability of molecular analysis, no studies to date have evaluated the transcriptional activation domain (exon 1) of the AR gene in this population of infertile men. Our study found no gross AR mutations in the individuals studied. These results emphasize the importance of further studies needed to understand the regulation of spermatogenesis.

Astrocytes modulate retinal vasculogenesis: effects on fibronectin expression.

Vasculogenesis is the formation of blood-vessels by differentiation of vascular precursor cells. Experiments using retinal models were designed to test the hypothesis that astrocytes influence this process by effects on the composition of the extracellular matrix. Retinal vasculogenesis was studied in relation to the migration of astrocytes and expression of the extracellular matrix proteins laminin and fibronectin by in vivo experiments in neonatal rats. The results show that astrocytes spread into the retina just ahead of the newly formed vessels, where they probably initiate vasculogenesis. They also establish that fibronectin, but not laminin, is expressed in the zone of vasculogenesis immediately prior to vessel formation. Increased amounts of fibronectin mRNA indicate that fibronectin is synthesized by cells within this same region during this same time period. Later, as the new vessels form, differentiation of endothelial cells is correlated with the appearance of pericytes in the vessel wall and laminin in the vascular basement membrane. In vitro experiments using conditioned medium approaches showed that astrocytes stimulate endothelial cell fibronectin expression. Taken together with the in vivo observations these in vitro results suggest that fibronectin expression is an essential component in the initiation of retinal vasculogenesis. This study is the first indication that astrocytes influence the fibronectin component of the extracellular matrix during retinal vasculogenesis and that expression of fibronectin precedes that of laminin in this process.

Cell specific distribution of LH/hCG receptor messenger ribonucleic acid in rat testicular Leydig cells.

By using four different cell isolation procedures, we previously identified two morphologically and biochemically distinct Leydig cell populations in rat testis. The light cells were vacuolated and bound 125I-labeled human choriogonadotropin (hCG) with high affinity but upon hCG stimulation in vitro, cAMP and testosterone production by these cells were minimal. On the other hand, the heavier cells displayed typical Leydig cell morphology and bound very little hCG but vigorously produced cAMP and testosterone (Browne, E.S., Bhalla, V.K., 1991, J. Androl. 12:132-139). This study examines the distribution of LH/hCG receptor mRNAs in the two cell types. The light cell fraction contains larger transcripts of LH/hCG receptor but the heavier Leydig cells contain shorter transcripts. The observations raises the intriguing possibility that shorter rather than larger LH/hCG receptor transcripts are responsible for the induction of a biologically functional, G-protein coupled, LH/hCG receptor in Leydig cells.

A unique point mutation in the androgen receptor gene in a family with complete androgen insensitivity syndrome.

OBJECTIVE: To further delineate the diversity of genetic alterations in the gene coding for the androgen receptor in individuals with the androgen insensitivity syndrome and to increase our understanding of the disease at the molecular level. DESIGN: This was a prospective study in which genomic deoxyribonucleic acid (DNA) from individuals with androgen insensitivity were examined through the polymerase chain reaction and DNA sequencing analysis. PATIENTS: Eleven complete and four individuals with partial androgen insensitivity syndrome were examined. RESULTS: Exons two through eight were grossly intact in all study subjects. Nucleotide sequence analysis revealed that three of three related family members with complete androgen insensitivity had the same guanine to adenine base substitution in exon five of the steroid-binding domain. CONCLUSION: The subsequent alanine to threonine amino acid conversion may have resulted in a configurational change of the androgen receptor protein leading to complete androgen insensitivity. This precise alteration has not been previously identified in the human androgen receptor gene in patients with the androgen insensitivity syndrome.

Cultured Müller cells have high levels of epidermal growth factor receptors.

High levels of epidermal growth factor (EGF)-receptors have been reported in membrane homogenates of bovine retinas, but the biologic function and tissue target of EGF in the retina have not been established fully. Because EGF participation has been suggested in the mechanisms of wound healing and Müller cells undergo changes after retinal injury, the authors studied EGF receptor expression and functional role of this substance in cultured Müller cells. These cells (isolated from normal rats) were tested for the glial cell markers: vimentin, S-100 protein, and carbonic anhydrase C. These markers were found to be positive through all passages used in the experiments. The 125I-EGF binding in Müller cells was highly specific, concentration dependent, and saturable. Compared with 3T3 fibroblasts, Müller cells bound threefold more EGF. Binding kinetics and Scatchard analyses showed the higher level of binding was related to the greater number of receptors on these cells (Müller cells, 2.4 x 10(5) receptors/cell; 3T3 fibroblasts, 7.1 x 10(4) receptors/cell) rather than a change in affinity of the receptors to bind the ligand. Nonlinear-regression analyses suggested the presence of two classes of affinity sites. The high level of EGF-receptor expression in Müller cells was confirmed by western blot analyses that showed increased reactivity of the approximately 170-kilodalton receptor band to a monoclonal anti-EGF receptor antibody. Moreover, EGF treatment of Müller cells resulted in two- to threefold increase in DNA synthesis, as evidenced by 3H-thymidine uptake studies. These findings support a functional role for EGF in Müller cell proliferation in retinal disease.

The presence of the testicular determining sequence, SRY, in 46,XY females with gonadal dysgenesis (Swyer syndrome).

Subjects with 46,XY gonadal dysgenesis (Swyer syndrome) have a distinctive phenotype. They are normal or tall in stature, lack somatic anomalies, and possess bilateral rudimentary gonads. Critical Yp deletions have been described in some cases, but in the majority no defects at the molecular level have been reported. To verify the presence or absence of SRY, the putative testicular-determining factor gene, specific primers were designed to amplify the conserved region of the SRY gene. Deoxyribonucleic acid from control males (n = 10) and sex-reversed females with the Swyer syndrome phenotype (n = 5) generated the anticipated 310 bp band. This Y-specific band was absent in the deoxyribonucleic acid from control females (n = 9). To search for possible point mutations, the amplified products of all study subjects and one control male were sequenced in both orientations. The base pair sequences were all identical and similar to the previously published report.

2,8-Dihydroxyadenine lithiasis in a Japanese patient heterozygous at the adenine phosphoribosyltransferase locus.

All reported cases of 2,8-dihydroxyadenine (DHA) lithiasis have been due to functional homozygous deficiency of adenine phosphoribosyltransferase (APRT). Here we describe the first case of DHA lithiasis in a patient who has functional APRT activity in cultured lymphoblasts. The patient is heterozygous for Japanese-type (type II) APRT deficiency as demonstrated by starch-gel electrophoresis and DNA sequence analysis. We also demonstrate the use of starch-gel electrophoresis for differentiation between the type II mutant enzyme and the wild-type enzyme.

Nerve growth factor promotes CNS cholinergic axonal regeneration into acellular peripheral nerve grafts.

Peripheral nerve grafts promote vigorous regeneration of adult mammalian CNS axons. Elimination of nerve-associated cells by freeze-thawing abolishes this promoting quality, possibly by creating inhibitory cellular debris and/or destroying the production of stimulatory factors by living Schwann or other cells. Here, debris-free acellular peripheral nerve segments placed between the disconnected septum and the hippocampal formation acquired almost no cholinergic axons after 1 month. However, such acellular nerve grafts treated before implantation with purified beta-nerve growth factor (NGF) contained nearly as many longitudinally oriented cholinergic axons as did fresh cellular nerve grafts. These results suggest that (i) NGF is required for the regeneration of adult CNS cholinergic axons into nerve grafts and (ii) an important function of living cells within peripheral nerve may be the production of neuronotrophic factors such as NGF.

Assessment of epidermal growth factor in the healing process of clean full-thickness skin wounds.

Epidermal growth factor is a potent stimulant of epithelialization. However, the usefulness of topical applications of epidermal growth factor in accelerating wound healing in full-thickness skin wounds with a large panniculus adiposus has not been clear. Four full-thickness skin incisions were made in the back of 10 female pigs that treated twice a day for 14 days with 2 ml of epidermal growth factor (300 ng/ml) or 2 ml of Ringer's lactate solution in a single-blind, randomized fashion. Two pigs received only epidermal growth factor, two pigs received only Ringer's lactate solution, and six pigs were treated with both solutions. The original skin plug was weighed to ensure similarity of groups. Photographs and measurements of each incision were taken every 7 days. The mean surface areas of the incisions treated with epidermal growth factor were 8.45, 7.50, and 2.30 cm2; in the incisions treated with Ringer's lactate solution the measurements were 8.42, 8.16, and 2.37 cm2 on observation days 1, 7, and 14, respectively. Although a trend toward a faster healing rate was noted in the incisions treated with epidermal growth factor, this difference was not statistically significant. With the doses and the time interval used between treatments, minimal benefit was obtained with epidermal growth factor when compared with Ringer's lactate solution.

Variant of A431 cells isolated by ricin A-conjugated monoclonal antibody directed to EGF receptor: phosphorylation of EGF receptor and phosphatidylinositol.

A monoclonal antibody specific for human EGF receptors was cross-linked to subunit A of toxic ricin. Using this conjugate, we isolated a variant of A431 cells, designated C1-B7, with approximately 40 times less EGF binding capacity. Unlike the parental cells, the C1-B7 variant was resistant to EGF-induced suppression of cell growth. The EGF receptors retained in this variant were of high-affinity type and susceptible to EGF-induced autophosphorylation. Membrane prepared from C1-B7 cells was highly phosphorylated in the presence of 2 microM [gamma-32P]-ATP, primarily on the lipid components shown as phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. This same level of lipid phosphorylation was observed on A431 membrane only in the presence of higher ATP concentrations. After addition of EGF to A431 membrane, phosphatidylinositol phosphorylation was significantly decreased with a concomitant increase in EGF-dependent protein phosphorylation. Thus, the EGF-dependent receptor-mediated protein phosphorylation precedes phosphatidylinositol phosphorylation. These observations support the idea that the growth inhibitory effect of EGF on A431 cells is caused by high ATP consumption due to the EGF-induced protein phosphorylation and reduction of phosphatidylinositol turnover.

Monoclonal antibody that immunoreacts with a subclass of human receptors for epidermal growth factor.

Spleen cells from BALBc mice immunized with human epidermoid carcinoma A431 cells were fused with mouse myeloma P3NP cells. One of the isolated hybridoma lines, B4G7, secreted a monoclonal antibody of the IgG class which inhibited the binding of [125I]-EGF to A431 cells and human fibroblasts, but not to mouse 3T3 cells. This inhibition was partial (65-70%) and Scatchard analysis of the EGF binding data suggested that the B4G7 antibody interacts preferentially with a low-affinity class of EGF receptors. This monoclonal antibody specifically precipitated EGF receptors (Mr = 170,000 and 155,000) of A431 cells which were directly crosslinked with [125I]-EGF. It also precipitated EGF receptors from cells whose surface proteins were labeled with 125I, from cells grown in the presence of [35S]-methionine or [32P]-orthophosphate, and from membrane fractions phosphorylated in vitro with [32P]-gamma-ATP. Receptors subjected to EGF-induced phosphorylation, both in vivo and in vitro, were also precipitated. The B4G7 antibody blocked approximately 70% of the EGF receptors in human fibroblasts, but did not stimulate DNA synthesis in these cells. However, in the presence of this antibody, cells showed the full mitogenic response to EGF, presumably through the unblocked receptors that are likely to be of the high-affinity type.

Genetic analysis of hyperproduction of epidermal growth factor receptors in human epidermoid carcinoma A431 cells.

Human epidermoid carcinoma A431 cells, possessing an extraordinarily high number of epidermal growth factor (EGF) receptors (1), were found to be hypotetraploid in their chromosome constitution and to contain two copies of intact chromosome 7 and two types of the translocation chromosomes involving chromosome 7 (M4 and M14) as well as several other rearranged chromosomes. The A431 cells were fused with mouse A9 cells, which lack EGF receptors (2) and are deficient in hypoxanthine phosphoribosyltransferase (3), and the human-mouse cell hybrid (AA series) were selected in HAT/ouabain medium (3, 4). The expression of high EGF binding ability was correlated with the presence of human translocation chromosome M4. AA hybrid clones that contained intact human chromosome 7 but not the marker chromosome M4 expressed only ordinary levels of EGF receptors. The EGF receptors expressed in the AA hybrids were proven to be of human nature by immunoprecipitation of the receptors cross-linked with [125I]EGF. These observations and our previous gene assignment of the EGF receptor to human chromosome 7 (2, 5) suggest that the marker chromosome M4 may carry an alteration(s) in the gene(s) involved in EGF receptor biosynthesis.

Genetics of receptors for bioactive polypeptides: expression of the human EGF receptor gene and internalization and processing of the receptor-bound EGF in human-mouse cell hybrids.

We previously postulated that the structural gene for epidermal growth factor (EGF) receptor is located on human chromosome 7 (1,2). In this study, EGF receptor and certain postreceptor functions were further analyzed in a unique cell hybrid line, C2B5, that retains only one human chromosome of an X;7 translocation besides a nearly complete mouse parental genome. Kinetics and Scatchard analysis of [125I]EGF binding to the C2B5 hybrid cells indicated that they carry a single class of EGF receptors with a dissociation constant of 4 x 10(-10) M. The receptors expressed in the hybrids are proven to be immunologically of human nature. The human EGF receptors now embedded in essentially mouse plasma membrane are subject to "down regulation" mediated by the ligand EGF. Analysis of the cell-bound EGF indicated that internalization and processing take place in the human-mouse cell hybrids. The degradation of EGF appears to be through a lysosomal pathway since it was substantially delayed or inhibited by lysosomotropic agents.

Genetics of cell surface receptors for bioactive polypeptides: binding of epidermal growth factor is associated with the presence of human chromosome 7 in human-mouse cell hybrids.

Mouse A9 cells, L-cell-derived mutants deficient in hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) were found to be incapable of binding (125)I-labeled epidermal growth factor (EGF) to the cell surface. The A9 cells were fused with human diploid fibroblasts (WI-38) possessing EGF-binding ability, and human-mouse cell hybrids (TA series) were isolated after hypoxanthine/aminopterin/thymidine/ouabain selection. Analyses of isozyme markers and chromosomes of four representative clones of TA hybrids indicated that the expression of EGF-binding ability is correlated with the presence of human chromosome 7 or 19. Four subclones were isolated from an EGF-binding-positive line, TA-4, and segregation of EGF-binding was found to be concordant with the expression of human mitochondrial malate dehydrogenase (MDHM; L-malate:NAD(+) oxidoreductase, EC 1.1.1.37), a marker for chromosome 7, but not with glucosephosphate isomerase (GPI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), a marker for chromosome 19. Furthermore, evidence from 27 clones of AUG hybrids that were produced between A9 and another human fibroblast line, GM1696, carrying an X/7 chromosome translocation indicated that EGF-binding ability segregates together with human MDHM and two X-linked markers, HPRT and glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP(+) 1-oxidoreductase, EC 1.1.1.49), that are located on the translocation chromosome 7p(+). These results permit assignment of the gene, designated EGFS, which is associated with the expression of EGF-binding ability, to human chromosome 7 and its localization to the p22-qter region. Because the EGF receptor is reported to be a glycoprotein the EGFS could be either a structural gene(s) for receptor protein or a gene(s) for modifying the receptor protein through glycosylation.