Two-subunit structure of the human thyrotropin receptor.

The extracellular and intracellular domains of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular alpha subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning beta subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5- to 3-fold excess of beta subunits over alpha subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane.     

A large intracellular pool of inactive Na channel alpha subunits in developing rat brain.

An intracellular pool of Na channel alpha subunits has been detected in developing brain cells in vivo and in vitro by phosphorylation with cAMP-dependent protein kinase, immunoprecipitation with specific antiserum, and NaDodSO4 gel electrophoresis or by radioimmunoassay. These alpha subunits are membrane-bound, contain complex carbohydrate chains, and have an apparent molecular weight of 260,000 like mature alpha subunits. In contrast to mature alpha subunits, the intracellular subunits are not covalently attached to a beta 2 subunit, and they do not bind saxitoxin with high affinity. They comprise 67-77% of the total immunoreactive alpha subunit in developing rat brain cells but are not a prominent component in the adult brain. It is proposed that this intracellular pool of alpha subunits forms a ready reserve of preformed subunits for incorporation into the surface membrane during periods of active membrane biogenesis. The results suggest that disulfide linkage of the alpha and beta 2 subunits, insertion into the cell surface membrane, and attainment of a functional conformation are closely related late events in the biogenesis of the Na channel. These processes may regulate the number of functional Na channels in the developing brain.     

mRNA-directed biosynthesis of α subunit of thyrotropin: Translation in cell-free and whole-cell systems

mRNA from mouse thyrotropic pituitary tumors was translated in frog oocytes (a whole-cell system) and in wheat germ extract and reticulocyte lysate (cell-free systems) in the presence of [(35)S]methionine. Synthesized peptides related to thyrotropin were identified in the three systems by immunoprecipitation with subunit-specific antisera developed against the alpha subunit of ovine lutropin (luteinizing hormone) and the beta subunit of bovine thyrotropin. In wheat germ extract and reticulocyte lysate, a single immunoprecipitable form of the alpha subunit of thyrotropin was synthesized with an apparent molecular weight of 14,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In the frog oocyte, three forms of immunoprecipitable alpha subunit of thyrotropin were synthesized with apparent molecular weights of 20,000, 14,000, and 10,000. The 20,000 form is similar to unlabeled rat pituitary standard alpha subunit and (35)S-labeled mouse tumor alpha subunit in cell cultures (20,000-21,000); thus, it may represent a precursor-cleaved and glycosylated form. The 14,000 form synthesized in all three systems probably represents the pre-alpha subunit of thyrotropin; the 10,000 form, synthesized only in the frog oocyte, could be a proteolytically cleaved but unglycosylated form. Because only the alpha subunit of thyrotropin was identified and no larger molecular weight immunoprecipitable form of either subunit was detected in any of the translation systems, alpha and beta subunits of thyrotropin appear to be translated from separate mRNAs.     

Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues.

The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the beta subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both alpha and beta subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the alpha and beta subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-linked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The alpha subunit of HGF contains four "kringle" structures, and the beta subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain.     

Functional reconstitution in Escherichia coli of the yeast mitochondrial matrix peptidase from its two inactive subunits.

The matrix processing peptidase from yeast (Saccharomyces cerevisiae) mitochondria was expressed in Escherichia coli via a plasmid-borne operon encoding the mature forms of the alpha and beta subunits of the enzyme. The subunits assembled into a fully active, soluble enzyme. The mature subunits were also expressed individually. The alpha subunit accumulated in large amounts and was obtained at a purity of 80% after a single chromatographic step. The beta-subunit-producing strain expressed an intact and a degraded form of the beta subunit, both of them soluble in the cytoplasm. Extract from either the alpha- or the beta-subunit-producing strain (S-alpha or S-beta extract, respectively), as well as the purified alpha subunit, was enzymatically inactive. However, precursor cleavage activity was restored by mixing either the S-alpha extract or the purified alpha subunit with the S-beta extract. The reconstituted processing activity was indistinguishable from the authentic holopeptidase.     

Biogenesis of the platelet receptor for fibrinogen: evidence for separate precursors for glycoproteins IIb and IIIa.

Congenital absence of platelet glycoproteins IIb and IIIa (GPIIb and GPIIIa) results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIb and GPIIIa are present as a heterodimeric, noncovalent complex in the platelet plasma membrane and function as the fibrinogen receptor. To characterize synthesis of these two proteins, RNA isolated from a human leukemia cell line that contains GPIIb and GPIIIa was translated in a wheat germ cell-free system. Polyclonal antibodies specific for each protein immunoprecipitated distinct [35S]methionine-labeled precursors, indicating that GPIIb and GPIIIa are translated from separate mRNAs. Moreover, using specific antibodies against either intact unreduced GPIIb or the beta subunit, we obtained evidence for synthesis of a common polypeptide precursor for GPIIb alpha and GPIIb beta. Based on experiments using microsomal membranes, it appears that GPIIb is integrated into the platelet membrane with little or no cytoplasmic component. These results suggest that precursors of GPIIb and GPIIIa may be encoded by separate genes and that each precursor is processed before delivery to the plasma membrane.     

Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor.

A discrepancy of about 20% exists between the molecular weight of the alpha subunit of Torpedo californica electroplax acetylcholine receptor as determined by gel electrophoresis of the mature protein (Mr 40,000 +/- 2000) and by nucleotide sequence analysis of cDNA (Mr approximately equal to 50,000). We demonstrate by amino acid sequence analysis that post-translational processing does not occur and that the mature subunit has a Mr of approximately equal to 50,000. The functional acetylcholine receptor contains two copies of this alpha subunit in addition to one each of related beta, gamma, and delta subunits. The binding sites for cholinergic ligands that are located on the alpha subunits have been shown to be nonequivalent. Amino acid sequence analysis of peptides obtained by proteolytic cleavage of the alpha subunit reveals that N-asparagine glycosylation at a single site (residue 141) occurs to a different extent in the two copies of this polypeptide in the mature protein and provides an explanation for nonequivalence of their binding sites.     

Expression of two forms of carp gonadotropin alpha subunit in insect cells by recombinant baculovirus.

There are two types of cDNA clones (designated alpha 1 and alpha 2) encoding the alpha subunit of carp gonadotropin. These two cDNAs are derived from different genes and encode proteins that differ by seven amino acid residues (three in the signal peptide and four in the mature polypeptide). Expression of these two cDNAs in insect cells by recombinant baculovirus revealed that the alpha 1 subunit, after noncovalent association with the beta subunit, has the same potency as the native alpha subunit purified from the pituitary. In contrast, the alpha 2 subunit can associate with the beta subunit, but only to form an inactive gonadotropin. Competition of the alpha 2 subunit with the alpha 1 subunit for association with the beta subunit decreases the gonadotropin activity of the alpha/beta complex. In addition, both alpha 1 and alpha 2 subunits are secreted into the culture medium by insect cells and have an apparent molecular mass approximately 5 kDa higher than that of the native alpha subunit. These results indicate that the insect cell-derived alpha 1 subunit is biologically active and that those four amino acid changes in the mature of alpha 2 protein affect the biological activity and thus provide valuable clues for the study of the structure-function relationship of the alpha subunit of glycoprotein hormones.     

The precursor region of a protein active in sperm-egg fusion contains a metalloprotease and a disintegrin domain: structural, functional, and evolutionary implications.

PH-30, a sperm surface protein involved in sperm-egg fusion, is composed of two subunits, alpha and beta, which are synthesized as precursors and processed, during sperm development, to yield the mature forms. The mature PH-30 alpha/beta complex resembles certain viral fusion proteins in membrane topology and predicted binding and fusion functions. Furthermore, the mature subunits are similar in sequence to each other and to a family of disintegrin domain-containing snake venom proteins. We report here the sequences of the PH-30 alpha and beta precursor regions. Their domain organizations are similar to each other and to precursors of snake venom metalloproteases and disintegrins. The alpha precursor region contains, from amino to carboxyl terminus, pro, metalloprotease, and disintegrin domains. The beta precursor region contains pro and metalloprotease domains. Residues diagnostic of a catalytically active metalloprotease are present in the alpha, but not the beta, precursor region. We propose that the active sites of the PH-30 alpha and snake venom metalloproteases are structurally similar to that of astacin. PH-30, acting through its metalloprotease and/or disintegrin domains, could be involved in sperm development as well as sperm-egg binding and fusion. Phylogenetic analysis indicates that PH-30 stems from a multidomain ancestral protein.     

Comparison of bovine and mouse pituitary glycoprotein hormone pre-α subunits synthesized in vitro

Poly(A)-containing RNA isolated from bovine and mouse pituitaries and a mouse pituitary thyrotropic tumor was translated in a wheat germ cell-free biosynthetic system. A precursor of the glycoprotein hormone alpha subunit, "pre-alpha," was immunoprecipitated from the translation mixtures with antiserum against bovine luteinizing hormone (LH; lutropin) alpha. The specificity of the immunoprecipitation was shown by competition with authentic bovine LHalpha and lack of competition with bovine thyroid-stimulating hormone (TSH; thyrotropin) beta. Bovine and mouse pre-alpha subunits migrated identically in sodium dodecyl sulfate gradient polyacrylamide slab gels with an apparent molecular weight of about 17,000. Pre-alpha comprised 2-3% and 20-30% of the total proteins translated with pituitary and pituitary tumor mRNA, respectively. Microanalysis of amino acid sequence of the pre-alpha subunits containing various radiolabeled amino acids gave the following partial sequence for mouse tumor pre-alpha: [Formula: see text] Met was also found in positions 1, 14, and 17 in mouse pituitary pre-alpha but only in residue 1 of the bovine pituitary pre-alpha subunit. Leu was found in identical positions in bovine pituitary pre-alpha, with an additional Leu in position 17. Leu in the common positions (12, 15, 19, and 22) has also been found in human choriogonadotropin pre-alpha subunit [Birken, S., Fetherston, J., Desmond, J., Canfield, R. & Boime, I. (1978) Biochem. Biophys. Res. Commun. 85, 1247-1253]. The data demonstrate that pituitary as well as placental glycoprotein hormone alpha subunits are synthesized with an amino-terminal hydrophobic extension, in accord with the "signal hypothesis" for secreted proteins. Furthermore, the positions of the hydrophobic amino acid Leu have been strictly conserved in pre-alpha subunits from various species and in two different tissues, the pituitary and placenta.     

Structural analysis and subunit interaction of insulin receptor from membranes of cultured embryonic chick heart cells

In previous studies of cultured embryonic chick heart, insulin hyperpolarized cells and slowed their beat rate through occupancy of a high affinity receptor. In the present studies, we chemically characterize the native structure and subunit interactions of this insulin receptor. A stokes radius of 87 A and an apparent molecular mass of 350,000 daltons were found for membrane proteins specifically cross-linked to [125I] insulin by disuccinimidyl suberate. A primary subunit of 125,000 daltons in dithiothreitol or 115,000 daltons in its absence (alpha-subunit) was heavily cross-linked. A smaller subunit had a size of 90,000 daltons (beta-subunit). This beta-subunit was not readily labeled by [125I]insulin cross-linking, but insulin enhanced 32P incorporation from [gamma-32P]ATP, enabling its visualization. Subunit interaction could be studied, because alkaline conditions produced dissociation of the native 350,000-dalton receptor. This spontaneous dissociation was not a result of proteolysis and was prevented by acid conditions, oxidants, or N-ethylmaleimide. Using alkaline conditions followed by chemical reduction in two-way gels, we directly visualized the native complex of 350,000 daltons dissociating into combinations of subunits of apparent sizes of 290,000 (alpha 2 beta), 220,000 (alpha alpha), and 195,000 (alpha beta) daltons. Dithiothreitol produced combinations of subunits, which differed from alkaline dissociation. In alkaline conditions, the 290,000 (alpha 2 beta) and 220,000 (alpha alpha) dalton combinations predominated, whereas dithiothreitol produced 190,000 (alpha beta)-dalton proteins, which suggested that alpha-S-S-alpha disulfide bonds existed and were susceptible to chemical reductants, while alpha-S-S-beta disulfide bonds were more sensitive to alkaline lysis. We conclude from these observations that the native insulin receptor of embryonic chick heart cell exists on the sarcolemmal membrane as a relatively homogeneous tetramer of nonhomologous subunits in an alpha 2 beta 2 configuration. The alpha-subunits are the primary sites for insulin binding, and a beta-subunit is autophosphorylated. alpha-S-S-alpha and alpha-S-S-beta bonding exist, and these disulfide bonds have different sensitivities to chemical reducing agents and alkaline lysis.     

Invariance and heterogeneity in the major structural and regulatory proteins of chick muscle cells revealed by two-dimensional gel electrophoresis.

A two-dimensional gel electrophoresis system is used to investigate some of the properties of desmin, the major subunit of the 100-A filaments from chick muscle cells, and to compare these properties to those of the other major contractile and regulatory proteins of muscle. Desmin from embryonic and adult smooth, skeletal, and cardiac muscle cells is resolved into two isoelectric variants, alpha and beta, which possess slightly different electrophoretic mobilities in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both the alpha and the beta variants from all six preparations appear to be identical in isoelectric point and apparent molecular weight. The alpha and beta desmin are present in approximately equal amounts in all three types of muscle, suggesting that both isoelectric variants of desmin serve as the structural subunits of the 100-A filaments in chick muscle cells. Tropomyosin also can be resolved into two subunits, alpha and beta, in all three types of muscle. However, in each type of muscle both subunits differ from their counterparts in the other types of muscle, either by molecular weight or by isoelectric point. These results indicate that, with regard to apparent isoelectric point and molecular weight, desmin, a major muscle structural protein, is invariant, while tropomyosin, a major muscle regulatory protein, exhibits heterogeneity in the three types of muscle.     

Identification of proteolytically resistant domains of human erythrocyte spectrin.

Digestion of purified human erthrocyte spectrin with proteolytic enzymes at 0 degrees C results in the production of intermediate-size peptides that resist further cleavage at 0 degrees C. By two-dimensional peptide analysis of these intermediate peptides it has been determined that five unique peptides are produced by tryptic cleavage of the alpha subunit of spectrin (band 1); these have apparent molecular weights of 80,000, 46,000, 46,000, 41,000, and 30,000 and account for 97% of the alpha subunit. Similarly, four unique peptides having apparent molecular weights of 74,000, 65,000, 33,000, and 38,000 account for 90% of the beta subunit (band 2). By examining larger peptide fragments, the linear alignment of the unique peptides along each of the spectrin subunits has been established. These results indicate that spectrin is composed of two nonidentical subunits, each containing multiple proteolytically resistant domains. These domains, which may be largely alpha-helical, seem to be connected by small protease-sensitive segments. The proteolytic resistance of these domains is not influenced by the multimeric state of the spectrin molecule.     

G-protein alpha o subunit: mutation of conserved cysteines identifies a subunit contact surface and alters GDP affinity.

The reversible association of alpha and beta gamma subunits of GTP-bindingproteins is important for signal transmission from a variety of cell-surface receptors to intracellulareffectors. Previous work showed that 1,6-bis(maleimido)hexane, which crosslinks cysteine residues, crosslinksalpha o and alpha i-1 to beta gamma. These crosslinks are likely to form through a conserved cysteinebecause 1,6-bis(maleimido)hexane can also crosslink alpha i-2, alpha 1, alpha s and Drosophila alpha1 to give products of the same apparent molecular weight as crosslinked alpha o beta gamma and alphai-1 beta gamma. These proteins have only two cysteines in common. Therefore, we mutated each of the twoconserved cysteines of alpha o to alanines. Mutation of Cys215 prevents crosslinking to beta gamma, butdoes not affect binding of guanosine 5'-[gamma-thio]triphosphate or the ability of the mutated alphasubunit to bind beta gamma. In models of the alpha subunit based on the crystal structure of p21ras,Cys215 is located on the face opposite to the GTP-binding site and near an area that changes conformationdepending on the nucleotide bound. This surface on the alpha subunit overlaps a putative effector bindingregion, raising important questions about the spatial organization of the proteins as they form ternarycomplexes. Mutation of Cys325 has no effect on crosslinking but, surprisingly, decreases by a factorof 10 the affinity of the mutated protein for GDP, relative to wild type, without changing the affinityfor guanosine 5'-[gamma-thio]triphosphate. This mutation falls within a region thought to contact receptorsand may represent a site through which receptors enhance the release of GDP.     

Direct observation of photolysis-induced tertiary structural changes in hemoglobin

Human Hb, an alpha2beta2 tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded R-state human Hb at cryogenic temperatures (25-35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the alpha subunit than in the beta subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state beta heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.     

Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.     

Structure of the mitochondrial F1 ATPase at 9-A resolution.

The soluble portion (F1 ATPase) of the mitochondrial ATP-synthesizing system is a multisubunit enzyme of molecular weight 380,000. It is composed of five different subunits, alpha, beta, gamma, and epsilon. The subunit stoichiometry is not known but there are strong suggestions that it is alpha 3 beta 3 gamma delta epsilon. We have determined the three-dimensional structure of the F1 ATPase of rat liver mitochondria to 9-A resolution by using x-ray diffraction techniques. The molecule appears to be formed by two equivalent halves, each formed by three regions of approximately equal size. These regions form a distorted hexagonal or octahedral arrangement. None of the regions form closed symmetrical trimers in the complex. It is proposed that, if the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, the major subunits exist in at least two different environments in the complex. In this arrangement, the different copies of the major subunits are functionally not equivalent. This observation appears to offer a natural explanation of the complicated binding and labeling data of F1 ATPases.     

The alpha subunit of the Na,K-ATPase specifically and stably associates into oligomers.

The Na,K-ATPase is a heterodimer consisting of an alpha and a beta subunit. Both Na,K-ATPase subunits are encoded by multigene families. Several isoforms for the alpha (alpha 1, alpha 2, and alpha 3) and beta (beta 1, beta 2, and beta 3) subunits have been identified. All these isoforms are capable of forming functionally active enzyme. Although there is general agreement that the Na,K-ATPase consists of alpha and beta subunits in equimolar amounts, the quaternary structure of the Na,K-ATPase and its functional significance is unknown. Several studies have demonstrated that the enzyme exists within the plasma membrane as an oligomer of alpha beta dimers. However, because the alpha beta protomer seems to be catalytically competent, the possibility exists that higher oligomers are irrelevant to function. The ability to express different alpha isoforms in insect cells and the availability of isoform-specific antibodies has provided the opportunity to test for the existence of stable and specific associations among alpha subunits. By coexpressing different alpha-subunit isoforms in cultured cells, we demonstrate that the Na,K-ATPase alpha subunits specifically and stably associate into oligomeric complexes. This same association among alpha-subunit isoforms was demonstrated in the native enzyme from rat brain. The interaction between Na,K-ATPase alpha subunits is highly specific. When the Na,K-ATPase alpha subunit is coexpressed with the alpha subunit from the H,K-ATPase, the H,K subunit does not associate with the Na,K subunit. Moreover, expression of the truncated alpha 1T isoform with the full-length alpha subunit demonstrates that the C-terminal portion of the polypeptide is important in the alpha-subunit association. Although these results do not clarify the functional role of alpha alpha associations, they do establish their highly specific nature and suggest that oligomerization of alpha beta protomers may be important to the stability and physiological regulation of the enzyme.     

Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells.

We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the alpha and beta subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 125I-labeled insulin but not insulin-like growth factor I, displays insulin-stimulated autophosphorylation of the beta subunit, and mediates insulin-stimulated 2-deoxyglucose uptake. We conclude that the human insulin receptor is synthesized, processed normally, and functional in this heterologous cell system.     

Rat Sertoli cell clusterin, alpha 2-macroglobulin, and testins: biosynthesis and differential regulation by germ cells.

Clusterin, alpha 2-macroglobulin and testins are three novel Sertoli cell proteins whose physiological functions may be related to cell-cell interactions in the seminiferous epithelium of the testis. We have demonstrated the biosynthesis of clusterin, alpha 2-macroglobulin, and testins by Sertoli cells in vitro using pulse-chase labeling analysis. For clusterin, two precursors with an apparent molecular weight (M(r)) of 72,000 (PH) and 66,000 (PL) were detected in the Sertoli cell cytosol in addition to the alpha (M(r) 43,000) and beta (M(r) 35,000) subunits of the mature protein. However, the precursors were not secreted into the medium since only the alpha and beta subunits of clusterin were detected. For alpha 2-macroglobulin and testins, no precursor molecules were detected either in the Sertoli cell cytosol or culture medium. The polarized secretory pattern of these proteins and their regulation by follicle stimulating hormone (FSH) and testosterone (T) were examined using a bicameral culture chamber that mimics the in vivo physiological conditions. Clusterin was secreted almost exclusively into the apical chamber of the bicameral culture unit with an apical:basal ratio of 30:1. In contrast, alpha 2-macroglobulin and testins had an apical:basal ratio of 1:1 and 1.5:1, respectively. Thus, the polarized secretory pattern for clusterin is different from alpha 2-macroglobulin and testins. It was noted that FSH and T, the known Sertoli cell regulators, did not affect the secretion of either clusterin or alpha 2-macroglobulin. Due to the morphological intimacy between Sertoli cells and germ cells in the adluminal compartment of the testis, the effects of germ cell-conditioned medium were investigated. Addition of germ cell-conditioned medium (1-30 micrograms protein) to the apical chamber of the bicameral culture unit caused a dose-dependent inhibition of clusterin and testins apical secretion and a slight but statistically significant stimulation of their basal secretion. In contrast, the secretion of alpha 2-macroglobulin by Sertoli cells was stimulated both apically and basally. These observations suggest that germ cell-conditioned medium contains a biological factor(s) that differentially regulates the bidirectional secretion of Sertoli cell proteins. These studies therefore reveal the complicated regulatory processes involved in cell-cell interactions in the seminiferous epithelium.