Rat brain membranes (BBM) bind (3-endorphin with high affinity and specificity. We report herein the identification of a high molecular weight ,-endorphin complex (receptor) in extracts of RBM preincubated with tritiated -endorphin, by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS). Upon isoelectric focusing, this complex gives a single peak with an isoelectric point (+SEM) of 4.50 ± 0.06. Sucrose density gradient experiments in H20 and 2H20 yield effective partial specific volume (v* = 0.814 cm3/g and sedimentation constant 820,w = 15.6 S. Gel filtration yields an estimate of the hydrodynamic radius of 73 A. The corresponding frictional ratio of 1.12 is consistent with an elliptical spheroid with an axial ratio of 3.1-3.2. The molecular mass of the complex is. estimated to be 690,000 daltons. The v of the complex is greater than that of CHAPS or most globular proteins. Solubilization of functional opiate receptors from brain or cultured neuroblastoma cells has been achieved with the use of a new class of detergent, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) (1, 2). We have found that [3H2]Tyr27-labeled human f3-endorphin (3H-/3h-EP) forms a complex with a component (receptor) of rat brain membrane (RBM) that survives solubilization with CHAPS and is sufficiently stable to permit physical characterization. MATERIALS AND METHODS (h-EP was prepared by solid-phase synthesis as described (3). H-Ph-EP was the product of catalytic reduction of a synthetic diiodotyrosine derivative of 3h-EP (4). CHAPS was synthesized -as described by Hjelmeland (5). Glycerol, bacitracin, and deuterium oxide were from Aldrich. Ampholines were from LKB and Pharmacia. Dithiothreitol, Hepes, and Tris were from Sigma. 3H20 was from Amersham. All other chemicals were of reagent grade. RBM were prepared as described (6) and stored at -70°C. Ten milliliters ofRBM in 50 mM Tris-HCI, pH 7.5/0.1% bacitracin/20% glycerol was incubated at room temperature for 1 hr with 1 nM of 3H-13h-EP, chilled on ice for 10 min, and centrifuged at 15,000 x g for 15 min. The pellet was resuspended in an equal volume of the same buffer; additions were made to give final concentrations as follows: dithiothreitol, 5 mM; Trasylol, 200 kallikrein inhibitor units/ml; phenylmethylsulfonyl fluoride, 0.1 mM; CHAPS, 10 mM. Insoluble material was removed by centrifugation at 100,000 X g for 1 hr. The supernatant was applied to a 2.5 x 30 cm column of Sepharose 6B equilibrated with 50 mM Tris-HCl (pH 7.5) containing 0.1% bacitracin and 1 mM CHAPS for isoelectric focusing experiments. For sucrose density gradient centrifugation, the eluant was 0.1 M KCI/10 mM Hepes/4 mM Tris-HCl/1 mM CHAPS (pH 7.5). The columns were eluted at 4°C with a peristaltic pump at a flow rate of 31 ml/hr. Radioactivity in each fraction was determined by liquid scintillation counting. The fractions containing the radioactive peak were pooled, decreased to 1-2 ml in volume by ultrafiltration on a Amicon YM-10 membrane filter, and used immediately or stored at -70°C. For isoelectric focusing, a 110-ml LKB isoelectric focusing column was used, with a 100-ml 5-50% sucrose gradient, 1% Ampholines, and 2 mM CHAPS. Electrode solutions were 0. 15 M phosphoric acid and 0. 15M NaOH. The sample ofsolubilized complex was added to the mixing chamber after 2/3 of the gradient had entered the column. Power was maintained at 1 W for 24 hr, and the temperature was 4°C. The gradient was collected in 1-ml fractions, the pH was read at room temperature, and the radioactivity was determined by liquid scintillation counting. For sucrose density gradient experiments, linear gradients (4.0 ml) were formed from 10-40% (wt/vol) sucrose in H20 or H20 containing 0.1 M KCI, 1 mM CHAPS, 10mM Hepes, and 4 mM Tris HCl (pH 7.5). Soluble complex or standards (100 IlI) were overlayed and centrifuged at 40,000 rpm in a Beckman SW 60 Ti rotor for 15 hr (H20) or 40 hr (2H20). Fractions of each gradient were collected from the bottom of the tube through a glass capillary and the refractive index of every other fraction was measured immediately. Radioactivity was determined after addition of 0.4 ml of 10% NaDodSO4. Protein of standards was estimated by Coomassie blue G-250 binding (7). The sedimentation constant (S20,w) and effective v3 (v*) of the 3-EP binding protein complex was determined by the method of Smigel and Fleischer (8). The FORTRAN program of Smigel (9) was rewritten for a small desktop computer in BASIC. To decrease the calculation time, a Simpson's rule numerical integration algorithm with adaptive step length (10) was substituted for that used by Smigel (9) and the viscosity and density gradients were interpolated with respect to radial distance by cubic splines (10). Buffer density was calculated from the t5 of its components, and buffer viscosity was measured in an Ostwald double-bulb viscometer at a number of temperatures. The ofCHAPS was determined with a mechanical oscillator densimeter. Preparative and analytic gel filtration on Sepharose 6B was used to estimate the hydrodynamic radius of the complex. Blue dextran and 3H20 were used to mark the void volume (V0) and internal volume (V.), respectively. The elution volume (Ve) of monomeric proteins of known hydrodynamic radius (11) was used to construct a calibration curve from the relation Re = A + B erf-1 (1-Kd), in which Kd = (Ve-V0)/(Vj-V0) (12). All chromatography experiments were carried out at 40C. Abbreviations: (-EP, 3-endorphin (subscript h, human); CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 3H-(3h-EP, [3H2]Tyr27-labeled human ,3-endorphin; RBM, rat brain membrane(s). 6494 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natd Acad. Sci. USA 79 (1982) 6495 Table 1. Binding of 3H-_Bh-EP to RBM before and after CHAPS solubilization 3H-Ph-EP, 0h-EP, Vo fractions, 25 pmol added pmol cpm

Rse, Axl, and c-Mer comprise a family of cell adhesion molecule-related tyrosine kinase receptors. Human Gas6 was recently shown to act as a ligand for both human Rse (Godowski et al., 1995) and human Axl (Varnum et al., 1995). Gas6 contains an NH2-terminal Gla domain followed by four epidermal growth factor-like repeats and tandem globular (G) domains, The G domains are related to those found in sex hormone-binding globulin and to those utilized by laminin and agrin for binding to the dystroglycan complex. A series of Gas6 variants were tested for their ability to bind to Rse and Axl. The Gla domain and epidermal growth factor-like repeats were not required for receptor binding, as deletion variants of Gas6 which lacked these domains bound to the extracellular domains of both Rse and Axl, A deletion variant of Gas6 containing just the G domain region was shown to activate Rse phosphorylation. These results provide evidence that G domains can act as signaling molecules by activating transmembrane receptor tyrosine kinases. Furthermore, they provide a structural link between the activation of cell adhesion related receptors and the control of cell growth and differentiation by the G domain-containing superfamily of proteins.

Another class of growth hormone (GH) secretagogues has been discovered by altering the backbone structure of a flexible linear GH-releasing peptide (GHRP). In vitro and in vivo characterization confirms these GH secretagogues as the most potent and smallest (M(r) < 500) reported. Anabolic efficacy is demonstrated in rodents with intermittent delivery. A convergent model of the bioactive conformation of GHRPs is developed and is supported by the NMR structure of a highly potent cyclic analog of GHRP-2. The model and functional data provide a logical framework for the further design of low-molecular weight secretagogues and illustrate the utility of an interdisciplinary approach to elucidating potential bound-state conformations of flexible peptide ligands.

Reexamination of the hexapeptide GH-releasing peptide (GHRP-6) structure/function has lead to the development of four novel classes of compound that stimulate GH release. Each class is represented as follows: a pentapeptide, G-7039; a tetrapeptide, G-7134; a pseudotripeptide, G-7502; and a rigid cyclic heptapeptide, G-7203. The EC50 values for these compounds, determined by GH dose-response curves using primary cultures of rat pituitary cells, were 0.18, 0.34, 10.6, and 0.43 nM, respectively. To demonstrate that these compounds were acting at the putative GHRP receptor, challenges were made using combinations that included GHRP-6 and GH-releasing hormone (GHRH). All four new classes further increased GH release in combination with GHRH, but not with GHRP-6. Homologous desensitization occurred after 45 min of exposure to the new compounds while the cells remained sensitive to GHRH. Somatostatin inhibited all of these compounds. Additionally, G-7039 elevated free calcium, as occurs with GHRP-6. All four classes elicited a robust GH release, a small increase in PRL, and no change in LH, FSH, ACTH, or TSH. We conclude that these novel compounds are potent and direct stimulators of pituitary GH release, with in vitro attributes that suggest mediation via a specific GHRP-like mechanism.

In many cell systems, cell-cell and cell-matrix interactions are mediated by integrins, a family of cell surface heterodimeric glycoprotein receptors. Osteoclast integrins may play a role in the process of bone resorption. Osteoclasts express the alpha v and beta 3 subunits of the vitronectin receptor and adhere to a wide range of proteins in vitro, all which contain the amino acid sequence Arg-Gly-Asp (RGD), an adhesion site recognition sequence common to many protein ligands that bind to integrins. The effect of kistrin, an RGD-containing snake venom protein, on osteoclast-mediated bone resorption was investigated in vivo and in vitro. When kistrin was infused into normocalcemic and hypercalcemic mice, serum calcium was significantly lowered at 3 and 6 h after the start of infusion, indicating an inhibitory effect on osteoclast activity in vivo. In vitro, kistrin potently inhibited bone resorption by isolated rat osteoclasts cultured on slices of bovine bone, and kistrin also inhibited the attachment of 293 cells expressing recombinant human alpha v beta 3 to fibrinogen (IC50 = 1 nM). These results indicate the potential therapeutic use of RGD-containing molecules for hypercalcemia of malignancy or for other disorders associated with bone loss.

Transforming growth factor‐β (TGF‐β) and bone morphogenetic protein 4 (BMP 4) are both able, under certain circumstances, to induce endochondral bone formation in vivo. This study compared the effects of TGF‐β and BMP 4 on the gene expression of a retinoic acid (RA) responsive rat clonal preosteoblast cell line, UMR 201, as well as the way in which these proteins interact with RA in these cells. Both similarities as well as differences between the effects and mechanism of action of TGF‐β1 and BMP 4 were demonstrated. TGF‐β1 (0.1 ng/ml) strongly induced matrix gla protein (MGP) mRNA and increased the steady state osteonectin (ON) mRNA level. Cotreatment with TGF‐β1 and RA did not result in a further increase in MGP mRNA expression. In contrast, BMP 4 alone had no influence on MGP or ON mRNA expression but it significantly enhanced the RA induction of MGP mRNA. Pro‐α(1) (l) collagen mRNA was increased by TGF‐β1 (1 ng/ml) and BMP 4 (50 ng/ml). The addition of either TGF‐β1 or BMP 4 together with RA resulted in a further increase in pro‐α1(l) collagen mRNA levels. Both RA and TGF‐β1, but not BMP 4, increased the transcriptional rate of the pro‐α 1(l) collagen gene. TGF‐β1 reduced the constitutive as well as RA‐induced expression of osteopontin (OP) mRNA while BMP 4 reduced only the constitutive expression of OP mRNA. RA increased the transcriptional rate of the OP gene. Since the responses of UMR 201 cells to these structurally related factors were not identical, the results lend support to the concept that the coordinated expression of members of the TGF‐β1 superfamily may be necessary to control the progression of specific cell types through their differentiation pathways. © 1993 Wiley‐Liss, Inc.

Osteogenin and related bone morphogenetic proteins are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and bone formation in vivo. The influence of osteogenin, purified from bovine bone, and of recombinant human bone morphogenetic protein-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor 1, 10 ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.

Interleukin-15 (IL-15) is a novel cytokine which shares activities and receptor components with IL-2. To investigate the biological roles of IL-15 in the human nervous system, we examined the expression of mRNAs for IL-15 and the IL-15 receptor three subunits (IL-15Rα, IL-2Rβ and IL-2Rγ) in human neural cell lines and tissues using reverse transcription–polymerase chain reaction and Southern blot analysis. The constitutive expression of high levels of IL-15 mRNA was observed in all the cell lines examined, including Y79 retinoblastoma, IMR-32 neuroblastoma, SK-N-SH neuroblastoma, U-373MG glioma, KG-1-C glioma, NTera2 teratocarcinoma and neurons derived from NTera2 cells following treatment with retinoic acid (RA). Among these cell lines, IL-15 protein was detectable at high levels in culture supernatants of SK-N-SH cells and NTera2-derived neurons. The expression of an alternatively-spliced transcript of the IL-15 gene was up-regulated in NTera2 cells during RA-induced neuronal differentiation, suggesting the existence of differentiation-dependent transcriptional regulation. The expression of IL-15 mRNA was also identifed in the human cerebral and cerebellar tissues, peripheral nerve and skeletal muscle, while the mRNAs for the complete set of IL-15R components were detectable only in U-373MG cells, cerebral and cerebellar tissues at significant levels. These results indicate that the expression of IL-15 but not of IL-15R mRNA is universal in human neural cell lines and tissues and raise the possibility that IL-15 acts as a neuroimmune regulatory factor in the human central nervous system.