The cytoskeletal protein talin contains at least two distinct vinculin binding domains

We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.     

Regionalization and redistribution of membrane phospholipids and cholesterol in mouse spermatozoa during in vitro capacitation

Fracture-label, surface-replica, and routine freeze-fracture techniques were used in combination with phospholipase A2-colloidal gold (PLA2-CG) and filipin as probes to study changes in the distribution of phospholipids and cholesterol, respectively, in morphologically defined plasma membrane domains of mouse spermatozoa during in vitro capacitation. In noncapacitated spermatozoa, quantitative analysis revealed that the fractured plasma membrane overlying the equatorial segment carried the highest PLA2-CG labeling density. The next highest labeling densities were found in the anterior acrosome region and the post-acrosomal region. On the external surface of the plasma membrane revealed by surface replicas, a uniform distribution of PLA2-CG was confined mainly to the acrosomal region of the head. The plasma membrane of the sperm tail had a relatively low labeling density for PLA2-CG. In freeze-fracture replicas of filipin-treated spermatozoa, the labeling density of filipin/sterol complexes (FSCs) was high in the plasma membrane over the acrosomal region where the FSCs were uniformly distributed. The postacrosomal region was weakly labeled. After in vitro capacitation, the densities of PLA2-CG and FSCs were significantly reduced in the fractured plasma membrane of the sperm head and the middle piece of the tail. However, surface replicas revealed an increased PLA2-CG labeling on the external surface of the plasma membrane covering the postacrosomal region, the middle piece, and the principal piece. Another major change detected in capacitated spermatozoa was the presence of small aggregates and patches of elevated, membrane-associated particles on the surface-replicated plasma membrane in the upper portion of the postacrosomal domain. Here the PLA2-CG labeling density was found to be higher than in noncapacitated spermatozoa. These results provide new information with respect to the reorganization and redistribution of phospholipids in specific regions of the plasma membrane during capacitation and provide further support for the concept that removal or loss of antifusigenic sterol from the sperm plasma membrane constitutes an important step of the capacitation process.     

Purification and characterization of protein D/E, a putative sperm-binding protein involved in fertilization

We describe a method for the efficient purification of a 32 Kd glycoprotein from rat epididymal tissue. The glycoprotein was purified by gel filtration, ion-exchange, affinity, and reverse phase high pressure liquid chromatography. The highly purified glycoprotein was radiolabeled with an iodinatable, cleavable, photoreactive cross-linking agent, 1-[N-(2-hydrox-5-azidobenzoyl)-2-aminoethyl]-4-(-hydroxysuccini mid yl)-succinate (HAHS). The soluble radiolabeled glycoprotein was bound to washed epididymal spermatozoa in a time-dependent, saturable, and reversible manner. Scatchard analysis demonstrates that there are approximately 3,403 binding sites/spermatozoon. The binding efficiency (Kd) for spermatozoa was approximately 2.0 x 10(-10) M. The function of this glycoprotein was verified by using an in vivo artificial insemination fertilization assay. The fertility rate for control spermatozoa was approximately 53%, but the rate for spermatozoa exposed to polyclonal anti-glycoprotein antibodies was only 5%. These data suggest that the binding of the glycoprotein to the surface of rat spermatozoa is mediated by a receptor-type mechanism and is involved in the fertilization process.     

Pig epidermal growth factor precursor contains segments that are highly conserved among species

The aim of the present work was to study the binding of [125I]-BLGA (beta-lactoglobulin variant A) to the plasma membrane fraction of hybrid cells. This binding increased as a function of time with on-rate and off-rate constant at 4.47 +/- 0.18 x 10(6) M-1 min-1 and 0.17 +/- 0.07 min-1, respectively (n = 3). The saturation study showed a single binding site type corresponding to a Kd at 8.26 +/- 2.98 nM and 14.02 +/- 2.61 x 10(12) sites per mg of the plasma membrane protein (n = 3). Competitive of binding BLGA was observed with BLGA, complexed with retinol and also with RBP (retinol-binding protein). Gel filtration of [125I]-BLGA incubated with Triton X-100 solubilized membrane showed the formation of a ligand-receptor complex. Cross-linking of the tracer to plasma membrane showed a complex with a M(r) at 69 kDa, suggesting a receptor M(r) of 51 kDa, as seen by autoradiography of SDS-PAGE.     

Nephron localization of Na/SO4(2-)-cotransport-related mRNA and protein

Previous studies using proteolytic fragments and synthetic peptides have indicated that domain I of human polymeric immunoglobulin receptor (PIgR) is necessary for ligand binding. The expression in E. coli, and subsequent IgM-affinity purification of domain I of human PIgR is described. The recombinant domain I protein (rDI) was similar in structure to native SC domain I in that it bound specifically to MAb 6G11, an antibody which recognizes a critical portion of the PIg binding site in domain I. The biological activity of rDI was indicated by high affinity binding to PIgA (Kd = 1.6 x 10(-7) M) and IgM (Kd = 5.1 x 10(-7) M). Domain I of human SC is therefore sufficient for binding to PIg.     

Chicken liver contains a large quantity of a G-protein-linked neurotensin receptor

Using 125I-labeled neurotensin (NT), chicken liver was found to contain high affinity, G-protein-linked receptors directed specifically towards the bioactive C-terminal portion of NT. Binding was proportional to membrane and optimal at pH 7.5. The apparent Kd (approximately 91 pM) for this single class of binding sites was similar to Kds reported for the high-affinity components of NT binding to mammalian brain and intestinal membranes. However, the binding capacity (Bmax, approximately 2.3 pmol/mg) was 10-100 times higher than values reported for these mammalian tissues. Binding was inhibited by GTP analogues and by treatment with pertussis toxin but not by cholera toxin. Treatments with alkaline solutions, shown to inactivate G-proteins, decreased subsequent binding at pH 7.5. Whereas low concentrations of Mg2+ (optimum, approximately 0.5 mM) enhanced NT binding, concentrations of 5 mM and above were inhibitory. Cross-linking of 125I-labeled NT to liver membranes using glutaraldehyde specifically labeled two substances of approximately 52 and approximately 90 kDa, which could represent different binding proteins or complexes. These data demonstrate the presence in chicken liver of large amounts of high-affinity NT receptor(s) coupled to pertussis toxin-sensitive G-protein(s).     

Endocytosis of androgen-binding protein (ABP) by spermatogenic cells

To test whether Sertoli cell-secreted ABP could serve as steroid carrier to the germ cell (GC) lineage, radiolabeled ABP and SHBG and gold SHBG were used for binding studies and for internalization studies based on transmission electron microscope analyses and autoradiography of the radiolabeled samples. The data clearly showed that: (1) rat and human germ cells possess a single class of binding sites for rat ABP and human SHBG respectively (Kd of 0.78 and 0.56 nM); (2) 1.7 x 10(10) and 2.7 x 10(10) sites/mg protein was found in the corresponding plasma membrane preparations; (3) the receptor peak was eluted in the same position as dextran blue: 2000 kDa (M(r) = 2 x 106) for labeled rat ABP; (4) in the whole GC lineage, the labeled ligand was internalized through an endocytic pathway involving clathrin coated structures and the distribution was similar throughout the maturation step, however striking differences in the internalization rate were revealed with regard to the maturation step; and (5) this internalization occurred even in ligated seminiferous tubules, via the Sertoli cells cytoplasm. When isolated rat GC were incubated in the presence of ABP, a dose dependent increase in labeled secreted protein was observed for spermatocytes (50-250%) whereas ABP had no effect on spermatids. Addition of steroids and ABP caused a 200 and 50% increase in labeled secreted proteins for spermatocytes and spermatids respectively. 2-D SDS-PAGE analysis revealed that ABP alone increased the secretion of specific spermatocyte proteins whereas steroids in the presence of ABP resulted in the synthesis of new spermatocyte secreted proteins. Taken together these results strongly suggest that ABP may be required for spermatogenesis either as a steroid transmembrane carrier or on its own.     

Determination of perfluorocarboxylic acids by gas-liquid chromatography in rat tissues

Gastrin plays an important role in regulating gastric acid secretion and gastrointestinal mucosal growth but its cellular sites of action in man have not been determined. Using cryostat sections of gastric mucosal tissue we have identified (125I-gastrin binding followed by fixation-wet emulsion autoradiography) and characterized (125I-gastrin binding followed by counting) a gastrin receptor binding site in the human stomach. This site displayed binding characteristics similar to those observed in isolated cell systems: specifically, 125I-gastrin binding was rapid (t1/2 approximately 10 min at 37 degrees C), temperature-dependent (3.5 fold more radioligand bound at 22 degrees C than at 4 degrees C) and saturable. The binding of the radioligand was also tissue specific and was five-fold greater in the gastric body than in the gastric antrum and duodenum. In the autoradiographs, silver grains were localized only to parietal cells and not to other epithelial cell types. In the presence of 40 nM gastrin grains were no longer present over parietal cells demonstrating that these sites were both saturable and of high affinity. These data provide the first demonstration of gastrin binding sites (putative receptors) on parietal cells in the human stomach and suggest that gastrin acts directly on these cells to help regulate gastric acid secretion and/or mucosal growth.     

Megalin (gp330): a putative endocytic receptor for thyroglobulin (Tg)

Megalin (gp330) is a large glycoprotein receptor found mainly on a group of absorptive epithelial cells, including renal proximal tubule, epididymal and thyroid cells. Megalin has been shown to bind multiple, unrelated ligands, mainly in vitro, and to mediate endocytosis of ligandsin cultured cells. However, physiologic ligands of megalin are largely unknown. In the present study we have demonstrated that purified rat megalin binds rat thyroglobulin (Tg) in solid phase assays, with anestimated Kd of 9.2+/-0.6 nM. Binding was calcium dependent and was almost completely inhibited by excess Tg, by three megalin ligands - lactoferrin, lipoprotein lipase and apolipoprotein J- and by the receptor associated protein (RAP), which inhibits binding of all megalin ligands. Three anti-megalin antibodies partially inhibited Tg binding to megalin. 125I labeled Tg bound to megalin was released by EDTA and heparin; the released product was shown by SDS-PAGE and autoradiography to be 660 kD (dimeric) Tg. However, an immunoblotting experiment showed binding of megalin both to monomeric (330 kD) and dimeric Tg. We propose that megalin, which is known to mediate ligand endocytosis and is found on the apical surface of thyrocytes, may participate in the endocytosis of Tg from the colloid, a process that is required for hormone release from Tg.     

Pharmacological characterization of [3H]idazoxan, [3H]RX821002 and p-[125I]iodoclonidine binding to alpha 2-adrenoceptors in rat cerebral cortical membranes

Binding characteristics of alpha 2-adrenoceptors in rat cerebral cortical membranes were compared using the antagonist radioligands [3H]idazoxan, [3H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002), and the partial agonist radioligand [125I]2-[2,6-(dichloro-4-iodophenyl)imino]imidazoline ([125I]iodoclonidine). With [3H]RX821002 and alpha 2-adrenoceptor subtype-selective competitors, both alpha 2A/D- and alpha 2C-adrenoceptor subtypes were detected, suggesting rat cortical membranes contain approximately 90% alpha 2A/D-adrenoceptor subtype and 10% alpha 2C-adrenoceptor subtype. Only alpha 2A/D-adrenoceptors were detected with [3H]idazoxan and [125I]iodoclonidine. All three radioligands bound to a single high affinity site (Kd = 0.3-1.6 nM). However, the densities of sites labeled by [3H]idazoxan and [125I]iodoclonidine were 50% greater than the density labeled by [3H]RX821002, likely representing non-adrenoceptor binding sites. The density of [125I]iodoclonidine binding sites in glycylglycine buffer was similar to that labeled by [3H]RX821002. These results suggest that: (1) alpha 2A/D-adrenoceptors are the predominant subtype in rat cerebral cortex, (2) demonstrate that the small number of alpha 2C-adrenoceptors in this tissue can be detected using prazosin to displace [3H]RX821002 binding, and (3) non-adrenoceptor binding with [125I]iodoclonidine can be minimized with the use of glycylglycine buffer.     

Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization

Nicotinic acetylcholine receptor protein (nAChR) has been solubilized from rat cerebral cortices by extracting a crude membrane fraction with the nonionic detergent Triton X-100 (polyoxyethylene-p-t-octylphenol). The solubilized nAChR was partially purified by affinity chromatography (Naja naja siamensis alpha-toxin affinity arm, linked to Sepharose 4B) and characterized by binding of 125I-labeled alpha-bungarotoxin. The reaction of labeled toxin and nAChR appears to be second order with a rate constant (k1) equal to 0.38 X 10(5) M-1 S-1 at 20 degrees. The toxin-nAChR complex dissociates with a dissociation rate constant (k-1) of 1.23 X 10(-5) S-1 at 20 degrees (t 1/2 = 15.6 h). The kinetically determined dissociation constant (Kd) for the complex is 3.24 X 10(-10) M. A variety of cholinergic ligands were studied for their ability to inhibit binding of labeled toxin. The results indicate that the brain receptor is indeed nicotinic. The s20, w and v of the toxin-nAChR complex in 0.1% Triton were determined by velocity sedimentation in D2O and H2O sucrose gradients. The values are 12.9 S and 0.80 cm3 g-1. The Stokes radius of the complex determined by gel filtration equals 7.5 nm. The Mr of the complex calculated from the hydrodynamic parameters, and corrected for bound detergent, equals 357,000.     

Binding of enterostatin to the human neuroepithelioma cell line SK-N-MC

SK-N-MC cells were found to possess binding sites for enterostatin, a peptide with central effects on appetite and sympathetic activation of brown adipose tissue during high-fat feeding. Scatchard analyses of the binding indicated one high-affinity binding (Kd = 0.5-1.5 nM) and one low-affinity binding (Kd = 15-30 nM) for 3H-enterostatin (APGPR). 125I-YGGAPGPR showed similar binding parameters as for the low affinity binding of 3H-APGPR. Met-enkephalin and beta3-casomorphin1-5 were found to displace the binding of 3H-APGPR to the SK-N-MC cells. Affinity purification of solubilized cells revealed an APGPR-binding protein estimated to 53 kDa which may represent a distinct enterostatin receptor. Cross-linking of 125I-YGGAPGPR to intact cells labeled one major protein with the same molecular size. There was no binding of enterostatin to four other human neuroblastoma/neuroepithelioma cell lines, named IMR-92, LAN#5, NB-1 #14 and SH5-SY.     

Calcitonin receptors on neoplastic mononuclear cells cultured from a human giant-cell tumor of the sacrum

Saturable, specific, high-affinity calcitonin receptors were demonstrated in cultured neoplastic mononuclear spindle cells from a giant-cell tumor of the sacrum of a 38-year-old woman. The receptor was analyzed by autoradiography and 125I-calcitonin binding assay. Binding reversibility of 125I-calcitonin to the cells was not complete and the structural specificity was indicated by the inability of unrelated hormones to compete with calcitonin. The 24,000 receptors/cell and dissociation constant (Kd) of 8.0 x 10(-10) M, calculated from linear Scatchard plots, suggested the existence of a single class of calcitonin binding sites in the neoplastic mononuclear cells. Flow-cytometric analysis in the primary culture showed that mononuclear cells consisted of mononuclear round cells of monocyte/macrophage lineage, which express more calcitonin receptors than neoplastic mononuclear spindle cells. Administration of calcitonin caused morphological and physiological alterations, resulting in involutional or irregular cytoplasmic shapes and inhibition of DNA synthesis in neoplastic mononuclear cells accompanied by the escape phenomenon. Cells preincubated with calcitonin showed a decrease in 125I-calcitonin binding activity, which could account for the escape phenomenon. The decrease in 125I-calcitonin binding was rapid, but the recovery was not observed for 24 h after elimination of calcitonin. This decrease may be caused by the disappearance of residual receptors or by a decrease in calcitonin affinity. The calcitonin-induced morphological changes and the inhibition of DNA synthesis of cells were revealed to be mediated by calcitonin receptors.     

Nerve growth factor processing and trafficking events following TrkA-mediated endocytosis

We expressed the high affinity nerve growth factor receptor TrkA in Chinese hamster ovary (CHO) fibroblasts to study nerve growth factor (NGF) trafficking and processing events following receptor-mediated ligand internalization in a nonneuronal and p75 minus cell line. These stable clonal cell lines express approximately 2.5 x 10(5) TrkA receptors and bind 125I-NGF with high affinity (Kd = 4 x 10(-10) M). The TrkA receptors are autophosphorylated on tyrosine residues upon NGF stimulation and are capable of tyrosine phosphorylating downstream signaling molecules. The t1/2 of 125I-NGF internalization is 5 min, and the probability of an occupied TrkA receptor internalizing within 1 min at 37 C is 9.8%. By 2 h following endocytosis, less than 10% of internalized 125I-NGF is degraded, as determined by TCA precipitation. Thirty minutes following ligand endocytosis, endocytosed 125I-NGF is delivered back to the cell surface and released by the cell (retroendocytosis), possibly by remaining associated with recycling TrkA receptors. We measured the effect of acidification on 125I-NGF-TrkA association and found that, at pH 6, 40% of 125I-NGF remains bound. Thus, NGF may remain associated with the TrkA receptor at low pH conditions in the endosome and can thereby be targeted back to the plasma membrane for release by the cell. In conclusion: 1) TrkA, in the absence of p75, is fully capable of mediating 125I-NGF endocytosis; 2) internalized 125I-NGF is slowly and inefficiently degraded; 3) following internalization, 125I-NGF is retroendocytosed; and 4) the ability of 125I-NGF to remain receptor-associated during acidic conditions may provide a mechanism for its retroendocytosis via recycling TrkA vesicles.     

An approach for assaying benzodiazepine receptor binding with radioiodinated ligand: 125I-labeled diazepam derivative

[125I]2'-Iododiazepam (IDZ) was prepared and its application in a benzodiazepine receptor binding assay was studied. [125I]2'-IDZ binds to the rat cortical membrane with a high affinity (Kd, 0.66 nM). Various benzodiazepines showed competition with [125I]2'-IDZ for the binding sites in the rat cortical membrane, and the specificity of its binding correlated well with that of [3H]diazepam (r = 0.992, p < 0.001). These findings suggested that [125I]2'-IDZ binds to the same sites as [3H]diazepam and indicated that [125I]2'-IDZ can be used in a benzodiazepine receptor assay.     

Methodologic standards for diagnostic test research in pulmonary medicine

The presence of functional nongenomic progesterone (P) receptors in human spermatozoa has been investigated by equilibrium binding studies in intact spermatozoa, ligand blot and Western blot analysis of sperm lysates, as well as determination of the effects of the steroid on sperm intracellular Ca2+ concentrations. Binding experiments were performed using progesterone-11alpha-glucuronide-[125I]iodotyramine as tracer. Computer analysis of competition curves using different steroids as competitors indicated the presence of two distinct binding sites for P. The high affinity site (Kd in the nanomolar range) appears to be specific for P, whereas the low affinity one (Kd in the micromolar range) binds with equal affinity 11beta-hydroxyprogesterone (11betaOHP) and 17alpha-hydroxyprogesterone (17alphaOHP). A significant correlation exists among affinity constants (as determined by binding studies) and EC50 values for the effects of P, 11betaOHP, and 17alphaOHP on intracellular Ca2+ in fura-2-loaded spermatozoa, strongly indicating the involvement of P-binding sites in the biological effect of the steroid. In particular, dose-response curves for P were biphasic, with an EC50 in the nanomolar range and another in the micromolar range. Conversely, curves for 11betaOHP and 17alphaOHP were monophasic, with an EC50 just in the micromolar range. Ligand blot analysis of sperm total lysates performed with peroxidase-conjugated P revealed the presence of two binding proteins of 54 and 57 kDa that were specific for P. Indeed, peroxidase-conjugated P binding was blocked by the simultaneous presence of the unconjugated steroid. Using alpha c262 antibody, which is directed against the P-binding domain of genomic receptor, we detected two proteins of similar molecular mass (54 and 57 kDa), whereas using antibodies directed against the DNA-binding and N-terminal domains of the genomic P receptors, the two proteins were not detected. In addition, p54 and p57 appear to be mostly localized in sperm membranes and virtually absent in the cytoplasm. The involvement of these proteins in the biological effects of P is indicated by the strong inhibitory effect of alpha c262 on P-induced acrosome reaction of capacitated human spermatozoa.     

Hemarthrosis of the knee and bone contusion

The displacement of porcine [125I] insulin bound to rat and lamprey isolated hepatocytes with unlabeled lamprey and porcine insulins was investigated. Binding affinity of lamprey insulin for insulin receptor of rat was similar to that of porcine insulin. In contrast, the binding affinity of lamprey insulin for its own insulin receptor was higher than for a rat receptor. To determine the binding affinity constants of lamprey insulin receptor, the competition binding experiments were carried out on isolated lamprey hepatocytes using lamprey insulin as unlabeled ligand and tracer. The affinity of the same binding sites on lamprey hepatocytes was assessed in similar experiments but employing porcine insulin as unlabeled ligand and tracer. It was found that while Kd of low affinity binding sites on lamprey hepatocytes were similar for lamprey and porcine insulins, the Kd of high affinity binding sites was different: the displacement curve for lamprey insulin being shifted to the left as compared to the curve for porcine insulin. The number of high and low affinity binding sites, calculated independently in Scatchard plots, was equal. We conclude that the high affinity insulin binding sites of lamprey but not of rat hepatocytes reveal some species specificity in ligand-receptor interaction.     

Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein

By using receptor-associated protein (RAP) as an affinity target, an intrinsic factor-vitamin B12 (IF-B12)-binding renal epithelial protein of approximately 460 kDa was copurified together with the transcobalamin-B12-binding 600-kDa receptor, megalin. IF-B12 affinity chromatography of renal cortex membrane from rabbit and man yielded the same approximately 460-kDa protein. Binding studies including surface plasmon resonance analyses of the protein demonstrated a calcium-dependent and high affinity binding of IF-B12 to a site distinct from the RAP binding site. The high affinity binding of IF-B12 was dependent on complex formation with vitamin B12. Light and electron microscope autoradiography of rat renal cortex cryosections incubated directly with IF-57Co-B12 and rat proximal tubules microinjected in vivo with the radioligand demonstrated binding of the ligand to endocytic invaginations of proximal tubule membranes followed by endocytosis and targeting of vitamin B12 to lysosomes. Polyclonal antibodies recognizing the approximately 460-kDa receptor inhibited the uptake. Immunohistochemistry of kidney and intestine showed colocalization of the IF-B12 receptor and megalin in both tissues. In conclusion, we have identified the epithelial IF-B12-binding receptor as a approximately 460-kDa RAP-binding protein facilitating endocytosis.     

Presence and characteristics of receptors for [D-Trp6]luteinizing hormone releasing hormone and epidermal growth factor in human ovarian cancer

This study was undertaken to establish the presence and characteristics of receptors for [D-Trp6]LH-RH on the membranes of human ovarian cancer. Specific binding of [125I, D-Trp6]LH-RH was found in 29 of 37 (78.4%) ovarian cancers and in 6 of 11 (54.5%) non-malignant human ovaries. Ligand binding was dependent on time and plasma membrane concentration in a fashion expected of a peptide hormone. Saturation, kinetic and displacement data were consistent with the presence of a highly specific, single class of non-cooperative binding site. On the basis of receptors affinity, LH-RH-receptor-positive ovarian cancers could be divided into two groups: high affinity group (Kd=2.71 +/- 0.60 nM; Bmax=0.46 +/- 0.07 pmol/mg membrane protein) comprising 55% of tumors, and low affinity group (Kd=78.0 +/- 19.6 nM; Bmax=9.44 +/- 2.68 pmol/mg membrane protein) which included 45% of tumors. LH-RH antagonist Cetrorelix showed an affinity to LH-RH receptors on ovarian cancers 14 times higher than the agonist [D-Trp6]LH-RH. Using 125I-epidermal growth factor, specific high affinity receptors were also detected in membranes from 13 of 24 (54%) ovarian cancers and 5 of 11 (45%) non-malignant ovaries. The demonstration of LH-RH receptors in human ovarian cancers provides a rationale for the use of therapeutic approaches based on LH-RH analogues in this malignancy. The probable involvement of growth factors in the development of ovarian cancers suggests the merit of trying a combined therapy based on analogs of LH-RH and somatostatin for this carcinoma.