Fibronectin's cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix

Fibroblasts organize the modular cell-adhesive glycoprotein fibronectin into a highly structured pericellular matrix by poorly understood mechanisms. Previous studies implicated an amino-terminal domain in matrix assembly and suggested that fibronectin's cell-adhesive domain and the corresponding fibroblast receptor were not involved in this process. To further elucidate the fibronectin region(s) involved in matrix assembly, we mapped a library of proteolytic fragments and antibodies to various fibronectin domains. The fragments and antibodies were used to probe the role of fibronectin's amino-terminal and cell-adhesive domains in a fibroblast matrix assembly assay. We found that fibronectin fragments including the first 25-kDa sequence of fibronectin and antibodies to amino-terminal domains inhibited pericellular matrix assembly. Polyclonal antibodies to the 40-kDa collagen binding domain following the 25-kDa amino-terminal domain also inhibited matrix assembly. However, collagen binding is not required for matrix assembly as neither monoclonals blocking collagen binding nor purified collagen binding domains themselves inhibited matrix assembly. Therefore, the amino-terminal region of fibronectin contains a site important in matrix assembly, and most activity is present in the first 25-kDa of fibronectin. Fibronectin's cell-adhesive domain and the fibroblast receptor binding to this domain also play an important role in fibronectin matrix assembly. Apart from a monoclonal antibody to the amino-terminal domain, only monoclonal antibodies binding to fibronectin's cell-adhesive domain and inhibiting cell adhesion also inhibited matrix assembly. In addition a 105-kDa fragment containing the cell-adhesive domain inhibited matrix assembly. We conclude that at least two discrete and widely separated sites in fibronectin with different binding properties--the carboxyl-terminal fibroblast cell-adhesive domain and an amino-terminal matrix assembly domain localized primarily within the first 25 kDa--are required for fibronectin pericellular matrix assembly by fibroblasts. Fibronectin's cell-adhesive domain and its cell surface-receptor complex appear to be involved in the matrix assembly process prior to a step involving the amino-terminal domain. We believe that this step is likely to be the initiation of cell-associated fibronectin fibril formation by the fibronectin-adhesive-receptor complex.     

The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19-25% sequence identity to the gamma-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu1a. The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6, and 3.9 mM for these cations, respectively) was very similar to that of the wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mM, respectively). For the mGlu1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved in CaR as Ser-147 and Ser-170, respectively. Each of these residues was mutated to alanines and tested pharmacologically using the endogenous agonist Ca2+. CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca2+ (4-fold increase in EC50) and maximal response (79% of wild-type response). CaR-S170A showed no significant response to Ca2+ even at 50 mM concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological profiles almost identical to that of the wild-type receptor. These data demonstrate that Ser-170 and to some extent Ser-147 are involved in the Ca2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors.     

Altered rate of fibronectin matrix assembly by deletion of the first type III repeats

The assembly of fibronectin (FN) into a fibrillar matrix is a complex stepwise process that involves binding to integrin receptors as well as interactions between FN molecules. To follow the progression of matrix formation and determine the stages during which specific domains function, we have developed cell lines that lack an endogenous FN matrix but will form fibrils when provided with exogenous FN. Recombinant FNs (recFN) containing deletions of either the RGD cell- binding sequence (RGD-) or the first type III repeats (FN delta III1-7) including the III1 FN binding site were generated with the baculovirus insect cell expression system. After addition to cells, recFN matrix assembly was monitored by indirect immunofluorescence and by insolubility in the detergent deoxycholate (DOC). In the absence of any native FN, FN delta III1-7 was assembled into fibrils and was converted into DOC-insoluble matrix. This process could be inhibited by the amino- terminal 70 kD fragment of FN, showing that FN delta III1-7 follows an assembly pathway similar to FN. The progression of FN delta III1-7 assembly differed from native FN in that the recFN became DOC-insoluble more quickly. In contrast, RGD- recFNs were not formed into fibrils except when added in combination with native FN. These results show that the RGD sequence is essential for the initiation step but fibrils can form independently of the III1-7 modules. The altered rate of FN delta III1-7 assembly suggests that one function of the missing repeats might be to modulate an early stage of matrix formation.     

A novel imprinted gene, HYMAI, is located within an imprinted domain on human chromosome 6 containing ZAC

Transient neonatal diabetes mellitus (TNDM) is a rare disease characterized by intrauterine growth retardation, dehydration, and failure to thrive due to a lack of normal insulin secretion. This disease is associated with paternal uniparental disomy or paternal duplication of chromosome 6, suggesting that the causative gene(s) for TNDM is imprinted. Recently, Gardner et al. (1999, J. Med. Genet. 36: 192-196) proposed that a candidate gene for TNDM lies within chromosome 6q24.1-q24.3. To find human imprinted genes, we performed a database search for EST sequences that mapped to this region, followed by RT-PCR analysis using monochromosomal hybrid cells with a human chromosome 6 of defined parental origin. Here we report the identification of a novel imprinted gene, HYMAI. This gene exhibits differential DNA methylation between the two parental alleles at an adjacent CpG island and is expressed only from the paternal chromosome. A previously characterized imprinted gene, ZAC/LOT1, is located 70 kb downstream of HYMAI and is also expressed only from the paternal allele. In the pancreas, both genes are moderately expressed. HYMAI and ZAC/LOT1 are therefore candidate genes involved in TNDM. Furthermore, the human chromosome 6q24 region is syntenic to mouse chromosome 10 and represents a novel imprinted domain.     

The amino-terminal heptad repeats of the coiled-coil neck domain of pulmonary surfactant protein d are necessary for the assembly of trimeric subunits and dodecamers

Pulmonary surfactant protein D (SP-D), a lung host defense protein, is assembled as multimers of trimeric subunits. Trimerization of SP-D monomers is required for high affinity saccharide binding, and the oligomerization of trimers is required for many of its functions. A peptide containing the alpha-helical neck region can spontaneously trimerize in vitro. However, it is not known whether this sequence is necessary for the complete cellular assembly of disulfide-cross-linked, trimeric subunits and dodecamers. For the present studies, we synthesized mutant cDNAs with deletions or site-directed substitutions in the neck domain of rat SP-D, and examined the assembly of the newly synthesized proteins after transfection of CHO-K1 cells. The neck domain contains three "classical" heptad repeat motifs with leucine residues at the "d position," and a distinctive C-terminal repeat previously suggested to drive trimeric chain association. Deletion of the highly conserved core of the latter repeat (FSRYLKK) did not interfere with the secretion of dodecamers with lectin activity. By contrast, deletion of the entire neck domain or deletion of one or two amino-terminal repeats resulted in defective molecular assembly. The secreted proteins eluted in the position of monomers by gel filtration under nondenaturing conditions. In addition, the neck + carbohydrate recognition domain of SP-D was necessary and sufficient for the trimerization of a heterologous collagen sequence located amino-terminal to the trimeric coiled-coil. These studies provide strong evidence that the amino-terminal heptad repeats of the neck domain are necessary for the intracellular, trimeric association of SP-D monomers and for the assembly and secretion of functional dodecamers.     

Role of type III homology repeats in cell adhesive function within the cell-binding domain of fibronectin

Recombinant fibronectin (FN) fragments and their mutant proteins were produced to elucidate the role of type III homology repeats in cell adhesive activity within the cell-binding domain of FN. Cell adhesive activity of the 11.5-kDa fragment, the cell attachment site of the cell-binding domain, was less than 0.1% that of native FN despite the presence of the Arg-Gly-Asp-Ser sequence. The activity increased as type III homology repeats were added to the N terminus of the 11.5-kDa fragment, and a 52-kDa fragment with four additional type III repeats had almost the same activity of native FN. Deletion of Arg-Gly-Asp from the fully active fragments completely abolished the cell adhesive activity. Deletion of one or two repeats from the 52-kDa fragment affected the extent of the cell adhesive activity, the degree of the effect being inversely correlated with the distance of the deletion from the type III repeat containing Arg-Gly-Asp-Ser. Rearrangement of type III repeats caused much loss of activity. These results suggest that the number and kinds of type III repeats and their correct alignment rather than the putative synergistic site decide the extent of the specific cell adhesive activity.     

Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly

Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6(Gag) or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.     

Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition

In this work, we used two approaches to localize the 90-kDa heat shock protein (hsp90)-binding site within the hormone-binding domain of the glucocorticoid receptor. In the first approach, derivatives of the glucocorticoid receptor deleted for increasing portions of the COOH terminus were translated in rabbit reticulocyte lysate, and the [35S]methionine-labeled translation products were immunoadsorbed with the 8D3 monoclonal antibody against hsp90. The data suggest that a segment from amino acids 604 to 659 (mouse) of the receptor is required for hsp90 binding. We have recently shown that the internal deletion mutant of the mouse receptor (delta 574-632) binds hsp90, although the complex is somewhat unstable (Housley, P. R., Sanchez, E. R., Danielsen, M., Ringold, G. M., and Pratt, W. B. (1990) J. Biol. Chem. 265, 12778-12781). The two observations indicate that amino acids 574-659 are involved in forming a stable receptor-hsp90 complex and that region 632-659 is especially important. To test this hypothesis directly, we synthesized three peptides corresponding to segments in region 624-665 and three peptides spanning the highly conserved sequence at amino acids 582-617, and we then tested the ability of the peptides to compete for the association of hsp90 with the L cell glucocorticoid receptor. In this assay, the immunopurified hsp90-free mouse receptor is incubated with rabbit reticulocyte lysate, which directs the association of rabbit hsp90 with the mouse receptor, simultaneously converting the receptor to the steroid binding state. All three peptides spanning region 624-665 and a peptide corresponding to segment 587-606 inhibited both hsp90 association with the receptor and reconstitution of steroid binding capacity. The data from all of the approaches support a two-site model for the hsp90-binding site in which the critical contact site occurs in region 632-659, which contains a short proline-containing hydrophobic segment and adjacent dipole-plus-cysteine motif that are conserved among all of the hsp90-binding receptors in the superfamily. A second hsp90 contact site is predicted in region 574-632, which contains the only highly conserved amino acid sequence in the receptor superfamily outside of the DNA-binding domain.     

The binding site in {beta}2-glycoprotein I for ApoER2' on platelets is located in domain V

The antiphospholipid syndrome is caused by autoantibodies directed against beta(2)-glycoprotein I (beta(2)GPI). Dimerization of beta(2)GPI results in an increased platelet deposition to collagen. We found that apolipoprotein E receptor 2' (apoER2'), a member of the low density lipoprotein receptor family, is involved in activation of platelets by dimeric beta(2)GPI. To identify which domain of dimeric beta(2)GPI interacts with apoER2', we have constructed domain deletion mutants of dimeric beta(2)GPI, lacking domain I (DeltaI), II (DeltaII), or V (DeltaV), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. DeltaI and DeltaII prolonged the clotting time, as did full-length dimeric beta(2)GPI; DeltaV had no effect on the clotting time. Second, DeltaI and DeltaII bound to anionic PL, comparable with full-length dimeric beta(2)GPI. DeltaV and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric beta(2)GPI, DeltaI, or DeltaII (mean increase 150%) were added to whole blood. No increase was found with plasma beta(2)GPI, DeltaV, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric beta(2)GPI, DeltaI, DeltaII, and the W316S mutant can interact with apoER2' on platelets. DeltaV did not associate with apoER2'. We conclude that domain V is involved in both binding beta(2)GPI to anionic PL and in interaction with apoER2' and subsequent activation of platelets. The binding site in beta(2)GPI for interaction with apoER2' does not overlap with the hydrophobic insertion loop in domain V.     

Zinc binding site in PICK1 is dominantly located at the CPC motif of its PDZ domain

PICK1 ( p rotein i nteracting with C k inase 1) containing a PDZ domain, a BAR domain, and two short acidic regions is as an adaptor protein that plays an important role in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor trafficking, cell morphology and migration, as well as in some diseases such as cancer, schizophrenia and pain. To better understand the physiological function of PICK1, we expressed the recombinant PICK1 and its truncated mutants in E.coli, and measured their zinc binding properties by fluorescence and competition assay. It is shown that PICK1 has one Zn²⁺-binding site. The Zn²⁺-binding properties of PICK1 are not appreciably affected after the removal of BARC domain (involving BAR domain and C-terminal acidic region). Deleting the N-terminal acidic region of NPDZ domain (involving PDZ domain and N-terminal acidic region) in PICK1 impairs its Zn²⁺-binding capacity.The mutation of the CPC (Cys-Pro-Cys) motif in the PDZ domain of PICK1 abolishes the ability of Zn²⁺-binding. In addition, Zn²⁺ can enhance the lipid-binding ability of PDZ domain as observed in both protein-lipid overlay assay and fluorescence analysis. The results presented in this report suggested that Zn²⁺ plays a regulatory role in the trafficking of PICK1 from the cytoplasm to cell membrane.     

Cysteine 144 in the third transmembrane domain of the creatine transporter is located close to a substrate-binding site

All creatine transporters contain a cysteine residue (Cys(144)) in the third transmembrane domain that is not present in other members of the Na+,Cl(-)-dependent family of neurotransmitter transporters. Site-directed mutagenesis and reaction with methane thiosulfonates were used to investigate the importance of Cys(144) for transporter function. Replacement of Cys(144) with Ser did not significantly affect the kinetics or activity of the transporter, whereas a C144A mutant had a higher K(m) (0.33 compared with 0.18 mm). Substitution of Cys(144) with Leu gave a mutant with a 5-fold higher K(m) and a reduced specificity for substrate. Low concentrations of 2-aminoethyl methanethiosulfonate (MTSEA) resulted in rapid inactivation of the creatine transporter. The C144S mutant was resistant to inactivation, indicating that modification of Cys(144) was responsible for the loss of transport activity. Creatine and analogues that function as substrates of the creatine transporter were able to protect from MTSEA inactivation. Na+ and Cl(-) ions were not necessary for MTSEA inactivation, but Na+ was found to be important for creatine protection from inactivation. Our results indicate that cysteine 144 is close to the binding site or part of a permeation channel for creatine.     

Independent assembly and secretion of a dimeric adhesive domain of von Willebrand factor containing the glycoprotein Ib-binding site

von Willebrand factor (vWF) is a multimeric glycoprotein that supports platelet adhesion on thrombogenic surfaces as part of the normal hemostatic response to vascular injury. We have employed a domain-specific expression strategy to analyze the biosynthetic processing steps and minimum structural requirements for assembly of the platelet receptor glycoprotein Ib-binding domain of vWF. A chimeric cDNA that codes for the vWF signal peptide and a segment of vWF internal primary sequence, residues 441-730, directs the secretion of a functional vWF fragment from mammalian cells. The recombinant molecule intrinsically assembles through intermolecular disulfide bond formation into a dimeric adhesive domain without contributions from other regions of vWF, including propeptide, previously indicated as essential for vWF multimer assembly. Prevention of N-linked glycosylation on the recombinant domain does not impair dimer formation or the ability to support platelet aggregation. These results identify a minimum structural element for vWF subunit assembly and provide new insights into the processing steps to produce vWF multimers and adhesive domains.     

A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions.

The functional configuration of Mu transposase (A protein) is its tetrameric form. We present here a model for the organization of a functional Mu A tetramer. Within the tetramer, assembly of each of the two active sites for Mu end cleavage requires amino acid contributions from the central and C-terminal domains (domains II and III respectively) of at least two Mu A monomers in a trans configuration. The Mu enhancer is likely to function in this assembly process by specifying the two monomers that provide their C-terminal domains for strand cleavage. The Mu B protein is not required in this step. Each of the two active sites for the strand transfer reaction is also organized by domain sharing (but in the reverse mode) between Mu A monomers; i.e. a donor of domain II (also the recipient of domain III) during cleavage is a recipient of domain II (and the donor of domain III) during strand transfer. The function of the Mu B protein (which is required at the strand transfer step) and that of the enhancer element may be analogous in that their interactions with Mu A (domain III and domain I alpha respectively) promote conformations of Mu A conducive to strand cleavage or strand transfer.     

Phylloquinone is the principal Mehler reaction site within photosystem I in high light

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy, and a part of the energy absorbed is lost in the reduction of O₂ to produce the superoxide radical (O2•−) via the Mehler reaction, which occurs principally within photosystem I (PSI). For decades, O₂ reduction within PSI was assumed to take place solely in the distal iron–sulfur clusters rather than within the two asymmetrical cofactor branches. Here, we demonstrate that under high irradiance, O₂ photoreduction by PSI primarily takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O₂ photoreduction rate constant in fully mature wild-type PSI from Chlamydomonas reinhardtii, complexes lacking iron–sulfur clusters, and a mutant PSI, in which phyllosemiquinone at the A-branch has a significantly longer lifetime. We suggest that the Mehler reaction at the phylloquinone site serves as a release valve under conditions where both the iron–sulfur clusters of PSI and the mobile ferredoxin pool are highly reduced.

In high light, O₂ reduction to superoxide radical primarily occurs at the phylloquinone cofactors of photosystem I rather than its terminal [4Fe–4S] clusters.     

Identification of critical regions within the TIR domain of IL-1 receptor type I

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC′ helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.     

Dual function for a unique site within the beta2I domain of integrin alphaMbeta2

Integrin activation has been postulated to occur in part via conformational changes in the I domain of the beta subunit (the betaI domain), especially near the F-alpha(7) loop, in response to "inside-out" signaling. However, direct evidence for a role of the F-alpha(7) loop in ligand binding and activity modulation is still lacking. Here, we report our finding that the F-alpha(7) loop (residues 344-358) within the beta(2)I domain has dual functions in ligand binding by alpha(M)beta(2). On the one hand, it supports intercellular adhesion molecule 1 (ICAM-1) binding to alpha(M)beta(2) directly as part of a recognition interface formed by five noncontiguous segments (Pro(192)-Glu(197), Asn(213)-Glu(220), Leu(225)-Leu(230), Ser(324)-Thr(329), and Glu(344)-Asp(348)) on the apex of the beta(2)I domain. On the other hand, it controls the open and closed conformation of the alpha(M)beta(2) receptor, thereby indirectly affecting alpha(M)beta(2) binding to other ligands. Switching the five constituent sequences of the ICAM-1-binding site within the beta(2)I domain to their beta(1) counterparts destroyed ICAM-1 binding but had no effect on the gross conformations of the receptor. Of the five ICAM-1 binding-defective mutants, four had normal or even stronger interaction with Fg and C3bi, as reported in our previous study. Synthetic peptides derived from the identified site inhibited alpha(M)beta(2)-ICAM-1 interaction and supported direct binding to ICAM-1. Most importantly, perturbation of the F-alpha(7) loop caused conformational changes within the beta(2)I domain, which was further propagated to other regions of alpha(M)beta(2). Altogether, our data demonstrate that inside-out signaling could modulate ligand binding directly by changing the ligand-binding pocket per se and/or indirectly by inducing multiple conformational changes within the receptor.