Sex hormone-binding globulin/androgen-binding protein: Steroid-binding and dimerization domains

Plasma sex hormone-binding globulin (SHBG) and testicular androgen-binding protein (ABP) are homodimeric glycoproteins that share the same primary structure, and differ only with respect to the types of oligosaccharides associated with them. The biological significance of these differences is not understood, but enzymatically deglycosylated SHBG and a non-glycosylated SHBG mutant both bind steroids normally. Various affinity-labelling experiments, and studies of recombinant SHBG mutants have indicated that a region encompassing and including Met-139 in human SHBG represents an important component of its steroid-binding site. Analyses of chimeric proteins comprising various portions of human SHBG and rat ABP have also indicated that residues important for the much higher affinity of human SHBG for steroid ligands are probably located within the N-terminal portion of these molecules. Recent studies of SHBG mutants have confirmed this, and a deletion mutant containing only the first 205 N-terminal residues of human SHBG has been produced which dimerizes and binds steroids appropriately. The introduction of amino-acid substitutions between Lys-134 and Phe-148 of SHBG has also indicated that residues including and immediately N-terminal of Met-139 may influence steroid-binding specificity, while those immediately C-terminal of Met-139 represent at least a part of the dimerization domain. These studies have also demonstrated that dimerization is induced by the presence of steroid ligand in the binding site, and that divalent cations play an important role in this process. Together, these data have led us to conclude that SHBG is a modular protein, which comprises an N-terminal steroid-binding and dimerization domain, and a C-terminal domain containing a highly-conserved consensus sequence for glycosylation that may be required for other biological activities, such as cell-surface recognition.     

The genes for SHBG/ABP and the SHBG-like region of vitamin K-dependent protein S have evolved from a common ancestral gene

Sex hormone binding globulin (SHBG) is the most important sex steroid transport protein in human plasma. It is the product of the same single gene as the androgen binding protein (ABP) of testis. Protein S is another protein, which is an important cofactor in the anticoagulation system and, as far as is known today, functionally unrelated to SHBG/ABP. Protein S also has a role in the complement system. A comparison of the human genes for SHBG/ABP and protein S reveals a sequence similarity, which is of a low grade only, between the SHBG/ABP protein and a similar sized COOH-terminal domain of protein S. However, the intron-exon organization exhibits a striking similarity in the two genes, illustrating evolutionary events leading to the appearance of two functionally different proteins from common ancestral genetic elements.     

Merosin promotes cell attachment and neurite outgrowth and is a component of the neurite-promoting factor of RN22 schwannoma cells.

The laminin-like protein merosin was purified from human placenta in intact form and as pepsin fragments and compared to laminin in heparin affinity chromatography and cell binding assays. Intact merosin and a small fragment of merosin comprising the last two repeats of the heavy chain g domain bind to heparin. Intact merosin and large pepsin fragments of merosin, but not the small C-terminal fragment, mediate the attachment and spreading of several types of cells and promote neurite outgrowth from neuronal cells similar to laminin and its corresponding fragments. Cells with various integrin-type receptors for laminin attached equally well to merosin and laminin, suggesting that several of the known laminin binding receptors also bind to merosin. Antibodies to the beta 1 subunit of integrins inhibited neurite outgrowth on merosin as well as on laminin, confirming the involvement of integrin-mediated interaction of cells with both merosin and laminin. Schwannoma cells, which have previously been shown to produce a laminin-like, neurite-promoting factor, synthesize merosin in vivo and in vitro as shown by protein and mRNA analysis. The results suggest that merosin, which is the more abundant basement membrane protein in the laminin family, has properties very similar to laminin despite differences in the structure of the heavy chain. Furthermore, merosin may be identical to or a component of the neurite-promoting factors previously reported from heart, muscle, and Schwann cells.     

Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits.

The distribution of subunits of the basement membrane proteins laminin and merosin in human and rabbit tissue was studied by immunofluorescence using monoclonal antibodies. The laminin A chain is present in epithelial, endothelial, and smooth muscle basement membranes. Merosin, as defined by its heavy chain M, is present in striated muscle and peripheral nerve. The A subunit colocalizes with at least two B subunits: B2 plus either B1 or the recently discovered B1 homologue S. The M subunit most often colocalizes with B1 and B2. Exceptions include the myotendinous junction, where M colocalizes with S, and the trophoblast basement membrane, where the M subunit colocalizes with S as well as B1. The presence of all five known subunits of the laminin family in placenta allowed isolation of their parent molecules in native form by the use of monoclonal antibodies in affinity chromatography. Four different heterotrimeric proteins could be identified: B1 chain-containing laminin (A-B1-B2), S chain-containing laminin (A-S-B2), B1-containing merosin (M-B1-B2), and S-containing merosin (M-S-B2). The data show that the proteins in the laminin family are heterotrimers composed of one heavy and two light chains; that most basement membranes contain predominantly one protein of the laminin family; and that laminin, as defined by the A subunit, has a much more restricted distribution than previously thought.     

SHBG region of the anticoagulant cofactor protein S: Secondary structure prediction,circular dichroism spectroscopy,and analysis of naturally occurring mutations

Protein S (PS) and growth arrest specific factor 6 (GAS6) are vitamin K-dependent proteins with similar structures. They are mosaic proteins possessing a carboxyl-terminal region presenting sequence similarity with plasma sex hormone binding globulin (plasma SHBG), although apparently not involved in steroid binding. The SHBG-like modules have sequence similarity with the G repeats of the chain A of laminin. Laminin G repeats have been reported to contain mainly β-strands (about 40–50%) but no or little α structure by circular dichroism (CD) spectroscopy. Secondary structure predictions carried out in the present work unexpectedly showed a 20 to 27% helices content in the SHBG region of PS/GAS6 (about 100 residues), while plasma SHBG and laminin G repeats had around 10% helices. CD measurements for human PS indicated also that its SHBG region had about 100 residues in α-helical structure. These data suggest that the SHBG region of PS/GAS6 on the one hand, and the laminin G repeats and possibly plasma SHBG on the other hand, could present important structural differences. Previously reported polymorphisms and point mutations leading to PS deficiency and thrombophilia have been analyzed with our structural predictions. We found a good agreement between these structural predictions, CD measurements, experimental and clinical data. This information allows us to gain insights into the three-dimensional structure of PS that will be helpful for the design of new experiments and future clinical investigations. Proteins 29:478–491, 1997. © 1997 Wiley-Liss, Inc.     

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 (K_d of 0.78 and 0.56 nM); (2) 1.7 × 10¹⁰ and 2.7 × 10¹⁰ 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 × 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.     

LG4–5 domains of laminin‐211 binds α‐Dystroglycan to allow myotube attachment and prevent anoikis

Poly(2‐hydroxyethyl methacrylate) (PolyHEMA) prevents cell attachment was used here to study anoikis, the process where cells die when unattached or attached to an inappropriate matrix, in mouse C₂C₁₂ myotubes. A method was developed to efficiently embed proteins into PolyHEMA and the effect on cultured myotubes was determined. Myotubes grown on PolyHEMA‐coated plates fail to attach to the surface and remain as rounded, suspended cells, undergo dramatic increases in apoptosis and necrosis, and the number of viable cells decreases. Incorporation of merosin (laminin‐211) or the short laminin globular (LG4–5) modules of the laminin‐α2 chain C‐terminus (called 2E3) that binds α‐dystroglycan diminishes both apoptosis and necrosis and increases viability while bovine serum albumin had a much lesser effect, showing the specificity of this effect for these matrix proteins. One sarcolemma receptor for laminin‐binding is α‐dystroglycan. An antibody which binds α‐dystroglycan but which does not block laminin‐binding (VIA4) had little effect on apoptosis or viability on merosin or 2E3 embedded plates while another antibody (IIH6) which specifically blocks binding dramatically decreased viability and increased apoptosis. When merosin or 2E3 are added to culture media rather than embedded on plates these can also increase viability and decrease apoptosis even though the cells remain in suspension, though the effect is not as great as found for the embedded proteins where the cells attach. Thus, we conclude that the binding of a small LG4–5 modules of laminin‐211 to α‐dystroglycan is important in preventing anoikis and that attachment plus binding is necessary for maximal cell survival. J. Cell. Physiol. 222:111–119, 2010. © 2009 Wiley‐Liss, Inc.     

Mammalian expression of the human sex steroid-binding protein of plasma (SBP or SHBG) and testis (ABP). Characterization of the recombinant protein.

A full-length 1,209 bp cDNA encoding the human sex steroid-binding protein of plasma (SBP or SHBG) and testis (ABP) was constructed and expressed in BHK-21 cells. The sequence agrees with the published gene and protein sequences. The cells were found to secrete SBP following transfection and G418r selection. The recombinant protein binds 5 alpha-dihydrotestosterone with a Kd of 0.28 nM. It also binds testosterone and 17 beta-estradiol but not progesterone, estrone or cortisol revealing a steroid-binding specificity identical to that of human SBP. SDS-PAGE patterns are less complex than human SBP and show a monomeric molecular weight of about 43 kDa.     

结构域B在鼠冠状病毒S蛋白的抗原性及膜融合中的作用

棘突(spike, S)蛋白是冠状病毒表面必不可少的跨膜糖蛋白,在病毒进入宿主细胞时具有结合受体和诱导膜融合的双重作用。大部分冠状病毒S蛋白的受体结合域位于S1-CTD(即相对应的结构域B),而经典的乙型冠状病毒模型鼠肝炎病毒(mouse hepatitis virus, MHV)的受体mCEACAM1a与S1-NTD(即相对应的结构域A)结合,其结构域B的作用仍未完全清楚。本研究通过构建结构域B和S2膜融合元件的缺失突变体,并使其在鼠神经母细胞瘤细胞系Neuro-2a内成功表达,证实了结构域B对病毒S蛋白导致的细胞-细胞间膜融合是必需的。用不同方法处理的病毒颗粒作为抗原免疫小鼠,所获得的多克隆抗体进一步显示,结构域B不但是S蛋白的主要抗原决定簇,而且能诱导中和抗体明显抑制病毒感染和S蛋白介导的膜融合作用。此结果为阐述不同冠状病毒的致病性与感染性差异提供了新思路。     

Isolation and partial characterization of high affinity laminin receptors in neural cells

Neural cells in culture (NG-108, PC12, chick dorsal root ganglion, chick spinal cord, and rat astrocytes) bind laminin with an apparent Kd of congruent to 10(-9) M. Laminin affinity chromatography of chick brain membranes washed with 150 mM NaCl and eluted with 0.2 M glycine buffer, pH 3.5, yields a single protein with an apparent molecular mass of 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing and peptide mapping indicate that the 67-kDa protein is distinct from bovine serum albumin (68 kDa) but indistinguishable from high affinity laminin receptors isolated from skeletal muscle. After electroblotting onto nitrocellulose paper and probing with 125I-laminin, this putative laminin receptor binds laminin specifically (100 ng/ml). A second protein (congruent to 120-140 kDa) is also detected with 125I-laminin (100 ng/ml) in the laminin affinity-purified membrane proteins. Both 67- and congruent to 120-140-kDa proteins can be laminin affinity-purified from cultures enriched for neurons (greater than 90%) following metabolic labeling with [35S]methionine. Our data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.     

Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.     

A Mr 80K hepatocyte surface protein(s) interacts with basement membrane components

We have identified a Mr 80K cell surface protein(s) from adult rat hepatocytes that binds basement membrane components, including collagen IV, heparan sulfate proteoglycan, and laminin. Freshly isolated hepatocytes were cell surface-labeled with 125I using the lactoperoxidase-catalyzed method, and detergent-solubilized membrane proteins were chromatographed on affinity columns prepared with purified basement membrane components. A Mr 80K protein was eluted with 0.15-1 M NaCl from a collagen IV column. Two proteins (Mr 80K and 38K) were eluted from a heparan sulfate proteoglycan column. The larger protein was also eluted from a column made with heparan sulfate side chains. Several proteins (Mr 80K, 67K, 45K, and 32K) bound to an affinity chromatography column made with the laminin A chain-derived synthetic peptide PA22-2, which is active for promoting cell attachment. When fractions eluted from these columns were analyzed by two-dimensional gel electrophoresis, the Mr 80K proteins showed similar patterns with a pI ranging from 8 to 9. The Mr 80K protein(s) may have an important role in the interaction of hepatocytes with basement membrane.     

Laminin A-related domains in crb protein of Drosophila and their possible role in epithelial polarization

It is shown that four domains of the integral membrane protein crb of Drosophila are homologous with five C-terminal domains of the laminin A chain and merosin. Since the latter domains of laminin A have been implicated in determining epithelial polarity, the homology may suggest a similar function for these domains of crb. Such a function would be consistent with the known importance of crb for organization of epithelia in Drosophila.     

Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor

The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379-16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL-metabolism, basement membrane assembly, calcium binding, and growth- and neurite-promoting activities.     

Identification of extracellular matrix ligands for the heparan sulfate proteoglycan agrin.

Agrin is a major brain heparan sulfate proteoglycan which is expressed in nearly all basal laminae and in early axonal pathways of the developing central nervous system. To further understand agrin's function during nervous system development, we have examined agrin's ability to interact with several heparin-binding extracellular matrix proteins. Our data show that agrin binds FGF-2 and thrombospondin by a heparan sulfate-dependent mechanism, merosin and laminin by both heparan sulfate-dependent and -independent mechanisms, and tenascin solely via agrin's protein core. Furthermore, agrin's heparan sulfate side chains encode a specificity in interactions with heparin-binding molecules since fibronectin and the cell adhesion molecule L1 do not bind agrin. Surface plasmon resonance studies (BIAcore) reveal a high affinity for agrin's interaction with FGF-2 and merosin (2.5 and 1.8 nM, respectively). Demonstrating a biological significance for these interactions, FGF-2, laminin, and tenascin copurify with immunopurified agrin and immunohistochemistry reveals a partial codistribution of agrin and its ECM ligands in the chick developing visual system. These studies and our previous studies, showing that merosin and NCAM also colocalize with agrin, provide evidence that agrin plays a crucial role in the function of the extracellular matrix and suggest a role for agrin in axon pathway development.     

Evidence that fibulin family members contribute to the steroid-dependent extravascular sequestration of sex hormone-binding globulin

Sex hormone-binding globulin (SHBG) binds steroids in the blood but is also present in the extravascular compartments of some tissues. Mice expressing a human SHBG transgene in the liver have human SHBG in their blood. In these animals, human SHBG accumulates within the stromal matrix of the endometrium and epididymis. This is remarkable because these tissues do not express the transgene. Human SHBG administered intravenously to wild-type mice in the presence of estradiol is rapidly sequestered within the endometrial stroma, and this prompted us to search for SHBG interacting proteins. Yeast two-hybrid screens revealed that fibulin-1D and fibulin-2 interact with the amino-terminal laminin G domain of SHBG. These interactions were verified in GST-pull down assays in which human SHBG bound the carboxyl-terminal domains of fibulin-1D and fibulin-2 in a steroid-dependent manner, with estradiol being the most effective ligand, and were enhanced by reducing the N-glycosylation of human SHBG. Like human SHBG, fibulin-1 and fibulin-2 concentrate within the endometrial stroma. In addition, SHBG co-immunoprecipitates with these fibulins in a proestrus uterine extract. These matrix-associated proteins may therefore sequester plasma SHBG within uterine stroma where it can control sex-steroid access to target cells. Given the interplay between fibulins and numerous proteins within the basal lamina, interactions between SHBG and matrix proteins may exert novel biological effects.     

Sex hormone-binding globulin receptor signal transduction proceeds via a G protein.

The plasma protein, sex hormone-binding globulin (SHBG) binds to a receptor (R(SHBG)) on cell membranes to form an SHBG-R(SHBG) complex. When an appropriate steroid binds to this complex, there is a rapid rise in intracellular cyclic adenosine monophosphate (cAMP). Although the system is moderately well characterized, the molecular cloning of R(SHBG) has not been accomplished and there is a paucity of evidence regarding the mechanism of transmission of the R(SHBG) signal. In this communication, we offer two independent lines of evidence that a G protein is involved in R(SHBG) signal propagation. Exposure of cell membranes containing R(SHBG) to a non-hydrolyzable analog of guanosine triphosphate (guanylyl-5'-imidodiphosphate) caused a substantive decrease in the binding of SHBG to R(SHBG). This behavior is typical of membrane receptors coupled to G proteins and has been used by others as evidence to support that relationship. Another set of experiments involved the assumption that, if R(SHBG)-induced increases in cAMP were diminished when the wild-type alpha subunit of a G protein was replaced with mutants that were inefficient/ineffective in signal transduction, then the idea that G proteins were involved in that signal would be buttressed. Hence, we infected COS-1 cells with a construct containing such mutants, along with a cAMP response element reporter, and demonstrated a marked decrease in R(SHBG)-engendered reporter activity, e.g. cAMP generation.     

Interaction of the low-molecular-mass, guanine-nucleotide-binding protein with the actin-binding protein and its modulation by the cAMP-dependent protein kinase in bovine platelets.

Platelets have been shown to possess several, different, low-molecular-mass, guanine-nucleotide-binding proteins (G-proteins) with molecular masses about 20-30 kDa. We report here that a 25-kDa G-protein copurified with the bovine platelet actin-binding protein (ABP), a cross-linker of actin filaments which is known to generate the three-dimensional network of actin. Both the G-protein and ABP were recovered in a fraction that was insoluble in Triton X-100 and were extracted in 0.6 M NaCl. Gel-filtration chromatography of the high-salt extract and rechromatography in a low-salt solution indicated that the two proteins may be associated with each other. The association of the two proteins was suggested by cosedimentation of the G-protein with the actin gel formed by actin and ABP. The amounts of the cosedimented G-protein and ABP was unaffected by guanosine-5'-O-[beta-thio]diphosphate and guanosine-5'-O-[gamma-thio]triphosphate, but the G-protein, not ABP, was partially released from the actin gel by phosphorylating ABP with cAMP-dependent protein kinase. Thus, the association of the two proteins was affected by modification of ABP, but not by modification of G-proteins. The physiological significance of the possible association of the two proteins might be that the membrane skeleton functions as a modulator of the G-protein, rather than that the G-protein modulates the function of the membrane skeleton which comprises ABP.     

Three-dimensional model of the SHBG-like region of anticoagulant protein S: new structure-function insights

Protein S (PS) is a vitamin K-dependent glycoprotein that consists of several modules including a C-terminal sex hormone-binding globulin (SHBG)-like domain that has been subdivided into two laminin LG-type domains. The SHBG-like region of PS is known to bind to a complement regulator molecule, C4b-binding protein (C4BP), coagulation factor Va (FVa) and receptor tyrosine kinases. Inherited PS deficiency has been associated with thromboembolic disease. Yet, study of the mechanisms by which the SHBG-like region of PS serves its essential functions has so far been hampered because of the lack of structural information. Recently, the three-dimensional (3D) structure of LG domains from plasma SHBG, laminin and neurexin have been reported and were found related to the pentraxin family. We used these X-ray structures to build homology models of the SHBG-like region of human PS. We then analyzed previously reported experimental/clinical data in the light of the predicted structures. A potential calcium-binding site is found in the first LG domain of PS and D292 could play a role in this process. This region is close to the interface between the two LG domains and is also surrounded by segments that have been suggested by synthetic peptide studies to be important for C4BP or FVa binding. The 39 point mutations linked to PS deficiencies or reported as neutral variants were rationalized in the 3D structure. Proteins 2001;43:203-216.