Functional characterization of the GDEP promoter and three enhancer elements in retinoblastoma and prostate cell lines.

GDEP (gene differentially expressed in prostate cancer aka. PCAN1), a newly discovered gene with remarkable tissue specificity, is a promising candidate for regulatory analysis because it exhibits a high level of expression that is limited to two tissues, the retina and the prostate. As these two tissues have different origins and disparate functions it is likely that the regulatory mechanisms responsible for expression are not shared in their entirety. In addition, both the retina and prostate are prime targets for gene therapy. To date there have been no functional studies of the GDEP promoter. Therefore to understand tissue-specific expression of GDEP we constructed promoter expression constructs. To further characterize functional regulatory regions within the GDEP gene, we investigated potential regulatory components for tissue-specific expression in the 40 kb intron of this gene. We have identified a 1.5 kb prostate-specific promoter from the proximal region of the GDEP gene. A smaller 0.5 kb promoter exhibited minimal activity in the retinoblastoma cell line Y79, but not in the prostate cells tested. In addition we have investigated three enhancer elements located in the 40 kb intron of the GDEP gene. We identified two enhancer elements that increase reporter gene expression in prostate cell line LNCaP and one additional enhancer element that increases expression in the Y79 cell line approximately 8-fold making it a strong retinal-specific enhancer.

Functional characterization of the HOXB13 promoter region.

Homeobox (HOX) genes are crucial regulators of cell growth and differentiation. These genes initiate and control gene expression cascades that drive development. More recently, the absent or aberrant expression of HOX genes has been implicated in cancer development. Despite the observance of these expression changes, the regulation of the HOX genes in adult tissues and how these genes become deregulated in cancerous tissues still needs much investigation. We characterized the promoter region of the HOXB13 gene. A 3 kb region upstream of the HOXB13 gene, which included the 5'UTR, increased reporter gene expression in LNCaP cells by approximately 99 fold over the promoterless control construct. A highly conserved 179 base pair fragment containing only the 5'UTR of the HOXB13 gene constituted a minimal promoter in the LNCaP cell line. Strong promoter activity was seen in the presence or absence of testosterone, although testosterone exposure did decrease expression in LNCaP cells by 50%. In an androgen insensitive cell line Du145, no sensitivity to testosterone was detected and a consistent low basal level of expression was observed. Since HOXB13 expression is highly tissue specific, we investigated the ability of the promoter to drive expression in tissues other than prostate. We observed highest expression in LNCaP cells with low levels of expression in lung, retinoblastoma, and colon cancer cells and higher expression in MCF7 breast cancer cells.

Role of transforming growth factor-beta in maintenance of function of cultured neonatal cardiac myocytes. Autocrine action and reversal of damaging effects of interleukin-1.

The three isoforms of transforming growth factor-beta (TGF-beta) have previously been implicated in embryonic development of the heart as well as in repair of myocardial damage after ischemia/reperfusion injury. TGF-beta 1 has also been localized intracellularly to both mitochondria and contractile filaments of cardiac myocytes, although its role in these structures has not been defined. We now report that exogenous TGF-beta stabilizes the beating rate of neonatal rat cardiac myocytes cultured on fibroblast matrix, and sustains their spontaneous rhythmic beating in serum-free medium. Moreover, using blocking antibodies to TGF-beta, we show that endogenous TGF-beta secreted by these myocytes acts in an autocrine fashion to maintain their beating rate. In contrast, IL-1 beta, an inflammatory mediator secreted by immune cells during myocardial injury, inhibits the beating of cardiac myocytes, and TGF-beta can overcome this inhibition. The antagonistic effects of TGF-beta and IL-1 were not observed when the myocytes were cultured on gelatin, as compared to native fibroblast matrix. The data indicate that TGF-beta is an important regulator of contractile function of the heart and have significant implications for understanding cardiac physiology in health and disease.

Phosphorylation is involved in transcriptional activation by the 1,25-dihydroxyvitamin D3 receptor.

The 1,25-dihydroxyvitamin D3 receptor becomes phosphorylated upon treatment with 1,25-dihydroxyvitamin D3. We have investigated the role of phosphorylation in the transcriptional activity induced by 1,25-dihydroxyvitamin D3 through its receptor. An active 1,25-dihydroxyvitamin D3-dependent transcription system was reconstituted in CV-1 cells by co-transfection of plasmids containing the rat 1,25-(OH)2D3 receptor DNA and a functional vitamin D response element (DRE) in a reporter gene construct. Treatment of these transiently transfected CV-1 cells with modulators of protein kinase A (8-Br-cAMP, PKIA and H-9) and phosphatases (Okadaic acid) resulted in mimicking or abolishing the transcriptional activity of 1,25-dihydroxyvitamin D3 in a receptor-dependent fashion. These modulators directly altered 1,25-dihydroxyvitamin D3 receptor phosphorylation. Therefore, the present results strongly suggest that phosphorylation plays a central role in the transcriptional activity of the 1,25-dihydroxyvitamin D3 receptor.

Breakpoint sequences of an 1;8 translocation in a family with Gilles de la Tourette syndrome.

Gilles de la Tourette syndrome (GTS) is a common, heritable neurological disorder manifested by chronic motor and vocal tics with childhood onset. Previous extensive linkage analysis failed to identify a GTS gene based on an autosomal dominant pattern of inheritance. Recently, a family was reported with a balanced chromosomal translocation t(1;8)(q21.1;q22.1) in family members with GTS or tics. Chromosome 8q22.1 was previously implicated in GTS by both association and linkage results. We therefore cloned and sequenced both translocation breakpoints from this family. The CBFA2T1 gene was identified 11 kb distal to the 8q22.1 breakpoint. Sequencing of CBFA2TI exons within 37 unrelated GTS patients failed to identify any mutations. However, it is possible that the translocation altered the expression of this gene or another nearby gene. Examination of the breakpoint sequences revealed a duplication of six nucleotides from chromosome 8 but no change in the chromosome 1 sequence. The sequences immediately flanking the breakpoints on the two chromosomes were modestly similar, but the breakpoints did not occur within known interspersed repeats. Our results add to our knowledge of the genetics of GTS and the mechanisms of balanced chromosomal translocations.

HIV-1 tat induced apoptosis of T-cells is not mediated by TGF-beta.

Recent reports have demonstrated that the HIV-1 transactivator protein, tat, induces apoptosis in T-lymphocyte cell lines, as well as in peripheral blood mononuclear cells, and stimulates a cascade of events resulting in up-regulation of the potent immunosuppressive cytokine, transforming growth factor-beta (TGF-beta). In this study we evaluated the ability of TGF-beta to mediate tat induced apoptosis in T-lymphocyte cell lines. T-cells treated exogenously with either TGF-beta1 or a combination of tat and pan-specific TGF-beta neutralizing antibodies showed little change in the amount of apoptosis. When treated with pan-specific TGF-beta neutralizing antibodies, Jurkat cells that stably express tat protein (Jurkat-tat ) showed only a modest decrease in apoptosis, while CEM-TART cells (CEM T-cells expressing both HIV-1 tat and rev ) demonstrated little change in the amount of apoptosis. In conclusion, we have demonstrated that TGF-beta does not play a significant role in mediating tat induced T-cell apoptosis.

Transforming growth factor-beta and multidrug resistance in chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) frequently respond to initial treatment, but then become resistant to chemotherapy. Studies have shown one important cause of chemotherapeutic resistance to be multidrug resistance (MDR). To investigate the potential role of MDR and transforming growth factor-beta (TFG-beta), a potent growth inhibitor of B lymphocytes, in the development of chemotherapeutic resistance in CLL, we evaluated 22 CLL patients for loss or mutation of TGF-beta receptors (TbetaR), plasma TGF-beta1 levels, and expression of MDR1 mRNA. Receptor crosslinking and immunoprecipitation experiments did not demonstrate loss of TbetaRs in any patients studied. No relationship between plasma TGF-beta1 levels and expression of MDR1 mRNA was seen. Correlation of plasma TGF-beta1 levels to disease stage revealed a consistent decline in plasma TGF-beta1 levels with advancing disease stage (P = 0.031).

Identification of a gene frequently mutated in prostate tumors.

Although prostate cancer is the second leading cause of cancer death for men in the United States, the genetics of tumor development are poorly understood. Several expressed sequence tagged genes (ESTs) that are expressed predominantly in the prostate have recently been identified, although their role in the development and maintenance of the prostate is unknown. Here, we demonstrate that the gene identified as UNIGENE cluster Hs. 104215, which codes for a message found predominantly in the prostate, may be important in tumor development. We name this gene PCan1 for Prostate Cancer gene 1. Northern blot experiments were performed using RNA isolated from tumor-derived cell lines and human prostate to determine the expression pattern of the gene. DNA sequencing was used to identify mutations that occurred in tumor tissue. By Northern blot analysis, this gene product was not detectable in LNCaP, DU 145, or PC-3 prostate cancer cell lines, although it was readily observed in RNA isolated from total prostate and from dissected central and peripheral regions of prostate. Sequence analysis of genomic DNA from LNCaP, DU 145, or PC-3 cells demonstrated a G/A polymorphism at position 193. Analysis of matched tumor-derived DNA and blood-derived DNA samples from 11 of 13 patients who had undergone a radical prostatectomy and who were homozygous for A in blood-derived DNA demonstrated mutation of position 193 in matched tumor samples resulting in G/A polymorphism. Sixteen additional patient samples were G/A polymorphic in both blood-derived DNA and tumor-derived DNA and two samples were GG in both blood-derived and tumor-derived DNA. Our results suggest that this gene may be a hot spot for mutation in prostate cancer, especially because our radiation hybrid mapping located this gene within a region identified in linkage mapping studies of affected families with prostate cancer. Loss of heterozygosity in prostate tumors has also been reported at the location of PCan1. Further studies to determine the functional role of this candidate tumor suppressor gene are warranted.

Expression and initial promoter characterization of PCAN1 in retinal tissue and prostate cell lines.

Prostate cancer is the most frequently diagnosed neoplasia in men and one of the leading causes of cancer-related deaths in men over 60. In an effort to understand the molecular events leading to prostate cancer, we have identified PCAN1 (prostate cancer gene 1) (also known as GDEP), a gene that is highly expressed in prostate epithelial tissue and frequently mutated in prostate tumors. Here we demonstrate its expression in neural retina, and retinoblastoma cell culture but not retinal pigment epithelial cell culture. We further characterize PCAN1 expression in the prostate cell lines RWPE1, RWPE2, and LnCAP FGC. We demonstrate an increase in expression when the cells are grown in the presence of Matrigel, an artificial extracellular basement membrane. Expression was time dependent, with expression observed on d 6 and little or no expression on d 12. Testosterone was not found to increase PCAN1 expression in this culture system. In addition, normal prostate epithelial cells co-cultured with normal prostate stromal cells did not exhibit PCAN1 expression at any time. To definitively locate the transcription initiation sites, we performed restriction-ligase-mediated 5' RACE, to selectively amplify only mRNA with a 5' cap. An initial characterization of the sequence upstream of the initiation sites determined six possible binding sites for the prostate specific regulatory protein NKX3.1 and four potential binding sites for the PPAR/RXR heterodimer that is involved in the control of cell differentiation and apoptosis.

Integration of genetic, clinical, and INR data to refine warfarin dosing.

Well-characterized genes that affect warfarin metabolism (cytochrome P450 (CYP) 2C9) and sensitivity (vitamin K epoxide reductase complex 1 (VKORC1)) explain one-third of the variability in therapeutic dose before the international normalized ratio (INR) is measured. To determine genotypic relevance after INR becomes available, we derived clinical and pharmacogenetic refinement algorithms on the basis of INR values (on day 4 or 5 of therapy), clinical factors, and genotype. After adjusting for INR, CYP2C9 and VKORC1 genotypes remained significant predictors (P < 0.001) of warfarin dose. The clinical algorithm had an R(2) of 48% (median absolute error (MAE): 7.0 mg/week) and the pharmacogenetic algorithm had an R(2) of 63% (MAE: 5.5 mg/week) in the derivation set (N = 969). In independent validation sets, the R(2) was 26-43% with the clinical algorithm and 42-58% when genotype was added (P = 0.002). After several days of therapy, a pharmacogenetic algorithm estimates the therapeutic warfarin dose more accurately than one using clinical factors and INR response alone.

An analysis of PAX1 in the development of vertebral malformations.

An analysis of PAX1 in the development of vertebral malformations. Due to the sporadic occurrence of congenital vertebral malformations, traditional linkage approaches to identify genes associated with human vertebral development are not possible. We therefore identified PAX1 as a candidate gene in vertebral malformations and congenital scoliosis due to its mutation in the undulated mouse. We performed DNA sequence analysis of the PAX1 gene in a series of 48 patients with congenital vertebral malformations, collectively spanning the entire vertebral column length. DNA sequence coding variants were identified in the heterozygous state in exon 4 in two male patients with thoracic vertebral malformations. One patient had T9 hypoplasia, T12 hemivertebrae and absent T10 pedicle, incomplete fusion of T7 posterior elements, ventricular septal defect, and polydactyly. This patient had a CCC (Pro)-->CTC (Leu) change at amino acid 410. This variant was not observed in 180 chromosomes tested in the National Institute of Environmental Health Sciences (NIEHS) single nucleotide polymorphism (SNP) database and occurred at a frequency of 0.3% in a diversity panel of 1066 human samples. The second patient had a T11 wedge vertebra and a missense mutation at amino acid 413 corresponding to CCA (Pro)-->CTA (Leu). This particular variant has been reported to occur in one of 164 chromosomes in the NIEHS SNP database and was found to occur with a similar frequency of 0.8% in a diversity panel of 1066 human samples. Although each patient's mother was clinically asymptomatic and heterozygous for the respective variant allele, the possibility that these sequence variants have clinical significance is not excluded.

Regulation of colony-stimulating factor-induced human myelopoiesis by transforming growth factor-beta isoforms.

Transforming growth factor-beta (TGF-beta) proteins are multifunctional regulators of cell growth and differentiation. The three isoforms, TGF-beta1, -beta2, -beta3 share approximately 70% identical amino acid sequence and are coded by three distinct genes. Growth and differentiation functions in which the isoforms have differential activity include: inhibition of colorectal cancer cell growth, migration of aortic endothelial cells, survival of ciliary ganglionic neurons, and binding to cell surface receptors. A previous paper reported that TGF-beta1 and TGF-beta2 had bimodal dose-dependent stimulatory and inhibitory effects on granulocyte-macrophage colony-stimulating factor induced Day 7 granulocyte-macrophage colony-forming units. The effects of TGF-beta3 were only inhibitory. At low concentrations, TGF-beta1 and -beta2 stimulated growth, whereas at higher concentrations both isoforms inhibited growth. We now report that TGF-beta1, TGF-beta2, and TGF-beta3 are similar to each other at low concentrations; at higher concentrations TGF-beta1 and TGF-beta3 inhibit growth, but TGF-beta2 stimulates growth. Our results are consistent with the known affinities of the TGF-beta isoforms with the Type II TGF-beta signaling receptor, which has greater affinity for TGF-beta1 and TGF-beta3 than TGF-beta2.

The cystic fibrosis conductance regulator gene exon sequence is normal in a patient with edematous eosinophilic nasal polyps.

Nasal polyps are the most common mass lesions found in the nose and their etiology is unknown. Nasal polyps from cystic fibrosis (CF) patients are histologically distinct from nasal polyps from patients without CF. It has been suggested that a mutation (G551D) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene may play a role in nasal polyp formation in patients without CF. To investigate the possibility that this or other CFTR gene exon mutations are required for nasal polyp formation, the CFTR gene exons were sequenced from peripheral blood DNA derived from an adult patient with edematous eosinophilic nasal polyps and no personal or family history of CF. No mutations or deletions were identified in any of the CFTR exons. A single polymorphism (A or G) was found in exon 10, base pair 1540, amino acid 470. This polymorphism was detected in 11 of 16 subjects (69%) with edematous eosinophilic nasal polyps and 10 of 21 normal subjects (48%) without nasal polyps and was not statistically significant (p = 0.316). These results demonstrate that mutations of the CFTR coding region are not a prerequisite for the formation of edematous eosinophilic nasal polyps.

Isolation and expression of rat 1,25-dihydroxyvitamin D3 receptor cDNA.

The cDNA for the 1,25-dihydroxyvitamin D3 receptor has been isolated by screening a rat kidney lambda gt11 expression library with a mixture of three previously identified anti-receptor monoclonal antibodies. Protein produced from this cDNA reacts with each of the three monoclonal antibodies but does not react with an antibody specific for the porcine receptor or an irrelevant antibody. In addition, the expressed protein specifically binds 1,25-dihydroxyvitamin D3 but does not bind estradiol, progesterone, testosterone, cortisol, or aldosterone. The nucleotide sequence for this cDNA has been determined and used to deduce the amino acid sequence of the protein. This amino acid sequence contains a region that matches perfectly the partial amino acid sequence determined for the receptor isolated from porcine intestine. This amino acid sequence also closely matches the reported amino acid sequence of the putative DNA-binding finger of the avian 1,25-dihydroxyvitamin D3 receptor.

Amino acids 67 and 68 of transforming growth factor-beta regulate binding to a glycosyl phosphatidyl inositol-linked membrane protein on vascular endothelial cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth and differentiation factor that affects almost all cells. Although equipotent in many cases, the three isoforms of TGF-beta (-beta1, -beta2, -beta3) have several important isoform specific activities. For example, TGF-beta2 binds with higher affinity to a 60 kDa cell-surface glycosyl phosphatidylinositol (GPI)-linked protein, expressed on vascular endothelial cells. We used chimeric TGF-beta proteins, in which selected regions of TGF-beta1 had been exchanged for the corresponding region of TGF-beta2, to demonstrate that amino acids 67 and 68 regulate binding of TGF-beta to this protein. Exchange of amino acids 67 and 68 of TGF-beta1 into TGF-beta2 resulted in a protein similar in affinity to TGF-beta1 for binding to the GPI-linked protein. In contrast, exchange of only amino acid 67 of TGF-beta1 into TGF-beta2, or exchange of only amino acid 68 of TGF-beta1 into TGF-beta2, resulted in a protein with affinity similar to that of TGF-beta2. This suggests that the coordinated change of Gln and His of TGF-beta1 to Thr and Ile at positions 67 and 68 alters the specificity of TGF-beta. Amino acids 67 and 68 are part of a surface-exposed alpha-helix that forms a projection away from the center of the TGF-beta molecule and is accessible for receptor binding.

Distinct functional domains of TGF-beta bind receptors on endothelial cells.

Transforming growth factor-beta (TGF-beta) is a multi-functional regulator of cell growth and differentiation. Three distinct isoforms of TGF-beta exist having similar, but not identical actions. TGF-beta 1, but not TGF-beta 2, binds to T beta RII and also to endoglin, a cell surface protein abundant on endothelial cells. In contrast, the affinity constant of TGF-beta 2 for alpha 2-macroglobulin is 10-fold greater than that of TGF-beta 1. TGF-beta 2 also binds better than TGF-beta 1 to a glycosyl phosphatidylinositol (GPI)-linked binding protein expressed on vascular endothelial cells. Using chimeric TGF-beta molecules, in which selected regions of TGF-beta 1 have been exchanged for the corresponding region of TGF-beta 2, we demonstrate here that amino acids 92-95 or 94-98 of TGF-beta determine isoform specific binding to endoglin. In contrast, exchange of only amino acids 95 and 98 did not alter TGF-beta specificity. Isoform specific binding to a GPI-linked protein on EJG endothelial cells was modulated by a region containing amino acids 40-68, although exchange of only amino acids 40-47 did not confer isoform specific binding. Significantly, the 92-98 region also modulates binding of TGF-beta to the type II receptor whereas isoform specific binding to alpha 2-macroglobulin requires concerted exchange of amino acids 45 and 47. Taken together, these results show that at least three different functional domains are important modulators of TGF-beta interaction with binding proteins and receptors.

Inhibition of growth by transforming growth factor-beta following fusion of two nonresponsive human carcinoma cell lines. Implication of the type II receptor in growth inhibitory responses.

Loss of growth regulation by transforming growth factor-beta (TGF-beta) may be an important step in carcinogenesis. We have used a cell fusion system to show that inhibition of growth by TGF-beta can be restored to carcinoma cell lines that are unresponsive to the inhibitory effects of TGF-beta. In a previous study, the EJ bladder carcinoma line was fused to the SW480 colon adenocarcinoma line and found to produce nontumorigenic hybrid cells along with one hybrid cell clone of low tumorigenicity. Here we show that the capacity of the nontumorigenic hybrid cells to respond to either TGF-beta 1 or TGF-beta 2 has been restored, while the parental or tumorigenic hybrid cells show little or no inhibition of growth following TGF-beta treatment. Cross-linking analyses with labeled TGF-beta 1 demonstrated much higher levels of the type II (85 kDa) receptor in the hybrid cells compared with the parental tumor lines. Both the parental and tumorigenic hybrid cell lines were capable of responding to TGF-beta as evidenced by increased levels of mRNA for fibronectin, type IV collagenase, and plasminogen activator inhibitor after treatment with TGF-beta 1. These results suggest that the type II receptor is necessary for mediating the effects of TGF-beta on inhibition of growth but not on gene activation of the hybrid cells.

Structure and regulation of the rat 1,25-dihydroxyvitamin D3 receptor.

Complementary DNA clones encoding the entire rat 1,25-dihydroxyvitamin D3 receptor were isolated, and the total 423-amino acid sequence was deduced. The amino acid sequence of the rat receptor is similar but not identical to the reported human receptor sequence. The cysteine-rich DNA-binding domain is completely conserved and the steroid-binding domain is greater than 93% conserved between rat and human. The cDNA was used as a probe in blot analysis of polyadenylylated RNA to show that the 1,25-dihydroxyvitamin D3 receptor mRNA is a single 4.4-kilobase mRNA that is expressed in intestine and kidney, slightly expressed in heart, and not detectable in liver and spleen. The receptor mRNA concentration is markedly increased during development of the rat intestine between day 14 and day 21, in accord with previous results obtained by measurement of receptor concentration by ligand binding or immunoblotting.

Transforming growth factor beta isoform 2-specific high affinity binding to native alpha 2-macroglobulin. Chimeras identify a sequence that determines affinity for native but not activated alpha 2-macroglobulin.

Transforming growth factor beta 2 (TGF-beta 2) is less potent than TGF-beta 1 in some endothelial cell proliferation assays due to the greater tendency of TGF-beta 2 to bind alpha 2-macroglobulin (alpha 2M). Substitution of TGF-beta 1 residues 40-47 into the TGF-beta 2 sequence yields a chimeric molecule that, like TGF-beta 1, expresses activity that is not substantially affected by serum alpha 2M (Burmester, J. K., Qian, S. W., Roberts, A. B., Huang, A., Amatayakul-Chantler, S., Suardet, L., Odartchenko, N., Madri, J. A., and Sporn, M. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8628-8632). In this investigation, we studied the binding of TGF-beta chimeras, which contain TGF-beta 1 residues 40-47, to both major conformations of human alpha 2M under apparent equilibrium conditions. Native alpha 2M, the primary form of this protein in serum, bound TGF-beta 2/beta 1 (40-82) and TGF-beta 2/beta 1 (40-47) with low affinity. The apparent KD values for the two chimeras and native alpha 2M were 310 and 330 nM, respectively. These values were much higher than the KD determined for TGF-beta 2 and native alpha 2M (11 nM) and equivalent to the KD determined for TGF-beta 1 and native alpha 2M. By contrast, both TGF-beta chimeras bound alpha 2M-methylamine, an altered conformation of alpha 2M, with high affinity (16 and 19 nM), which is characteristic of TGF-beta 2 and not TGF-beta 1. Fetal bovine heart endothelial cell DNA synthesis was inhibited to a similar degree by TGF-beta 1, TGF-beta 2, TGF-beta 2/beta 1 (40-82), and TGF-beta 2/beta 1 (40-47) in the presence of dilute (0.2%) fetal bovine serum. When 0.07 microM alpha 2M-methylamine was added, the activities of TGF-beta 2, TGF-beta 2/beta 1 (40-82), and TGF-beta 2/beta 1 (40-47) were significantly counteracted while the activity of TGF-beta 1 was unchanged, as would be predicted by the equilibrium binding analyses. These studies indicate that the TGF-beta structural elements, which mediate binding to native alpha 2M and conformationally transformed alpha 2M, are not equivalent. Residues 40-47 are critical in determining affinity for native alpha 2M but are less important in determining affinity for alpha 2M-methylamine.

Mutational analysis of a transforming growth factor-beta receptor binding site.

Transforming growth factor-beta s (TGF-beta 1, -beta 2, -beta 3) are important regulators of cell growth and differentiation which share approximately 70% identical amino acids. Using LS513 colorectal cells, which are growth inhibited by TGF-beta 1 (ED50 of 100 pM), but are refractory to TGF-beta 2 (ED50 of 50,000 to 100,000 pM), we have determined that amino acids 92-98 of TGF-beta specify growth inhibition. The chimeric protein TGF-beta 1/beta 2(92-98), in which amino acids 92-98 of TGF-beta 1 were exchanged for the corresponding amino acids of TGF-beta 2, was indistinguishable from TGF-beta 2 at inhibiting growth of LS513 cells. In contrast, both TGF-beta 1/beta 2(92-95) and TGF-beta 1/beta 2(94-98) inhibited the growth of LS513 cells with an ED50 of approximately 1000 pM. TGF-beta 1/beta 2(95-98), in which amino acids 95-98 of TGF-beta 1 have been replaced with the corresponding amino acids of TGF-beta 2, had full activity and was indistinguishable from TGF-beta 1. Receptor cross-linking experiments demonstrated that binding of the chimeras to the type I and type II receptors of LS513 cells was consistent with their biological activity. TGF-beta 1/beta 2(95-98), TGF-beta 1/beta 2(92-95) and TGF-beta 1/beta 2(94-98) were each similar to TGF-beta 2 in that they failed to bind to the soluble Type II receptor in a solid-phase assay. These results demonstrate that amino acids 92-98 are involved in the interaction between TGF-beta and its signaling receptors and they show that modest changes within this region can substantially alter biological response.