Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits

Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.     

Interaction of the tRNA(Phe) acceptor end with the synthetase involves a sequence common to yeast and Escherichia coli phenylalanyl-tRNA synthetases

Modified lysines resulting from the cross-linking of the 3' end of tRNA(Phe) to yeast phenylalanyl-tRNA synthetase (an enzyme with an alpha 2 beta 2 structure) have been characterized by sequencing the labeled chymotryptic peptides that were isolated by means of gel filtration and reversed-phase chromatography. The analysis showed that Lys131 and Lys436 in the alpha subunit are the target sites of periodate-oxidized tRNA(Phe). Mutant protein with a Lys----Asn substitution established that each lysine contributes to the binding of the tRNA but is not essential for catalysis. The major labeled lysine (K131) belongs to the sequence IALQDKL (residues 126-132), which shares three identities with the peptide sequence ADKL found around the tRNAox-labeled Lys61 in the large subunit of Escherichia coli phenylalanyl-tRNA synthetase [Hountondji, C., Schmitter, J. M., Beauvallet, C., & Blanquet, S. (1987) Biochemistry 26, 5433-5439].     

A molecular basis for integrin alphaMbeta 2 ligand binding promiscuity

The leukocyte integrin alpha(M)beta(2) is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of alpha(M)beta(2), the inter-integrin chimera was created. In the chimeric integrin, the betad-alpha5 loop-alpha5 helix segment comprised of residues Lys(245)-Arg(261) from the alpha(M)I domain of alpha(M)beta(2) was inserted into the framework of alpha(L)beta(2). The construct was expressed in HEK 293 cells, and the ability of generated cells to adhere to fibrinogen and its derivatives was characterized first. Grafting the alpha(M)(Lys(245)-Arg(261)) sequence converted alpha(L)beta(2) into a fibrinogen-binding protein capable of mediating efficient and specific adhesion similar to that of wild-type alpha(M)beta(2). Verifying a switch in the binding specificity of alpha(L)beta(2), the chimeric receptor became competent to support cell migration to fibrinogen. Mutations at positions Phe(246), Asp(254), and Pro(257) within Lys(245)-Arg(261) of alpha(M)beta(2) produced significant decreases in cell adhesion, illustrating the critical role of these residues in ligand binding. The insertion of alpha(M)(Lys(245)-Arg(261)) imparted to the chimeric integrin the ability to recognize many typical alpha(M)beta(2) protein ligands. Furthermore, cells expressing the chimeric receptor, but not alpha(L)beta(2), were able to stick to uncoated plastic, which represents the hallmark of wild-type alpha(M)beta(2). These results suggest that alpha(M)(Lys(245)-Arg(261)) serves as a consensus binding site for interaction with a variety of distinct molecules and, thus, may define the degenerate recognition properties inherent to alpha(M)beta(2).     

Cloning and expression of a divergent integrin subunit beta 8

Rabbit and human cDNA clones have been identified that encode a novel integrin beta subunit. The sequences that encode this subunit, which has been designated as beta 8, were isolated initially from rabbit placental cDNA libraries using an oligonucleotide probe derived from a highly conserved region of integrin beta subunit sequences. The rabbit clone was used to isolate human beta 8 cDNA clones from human placental and MG-63 osteosarcoma cell libraries. The putative beta 8 polypeptides, which comprise 769 and 768 residues in human and rabbit, respectively, show a high degree of inter-species conservation (approximately 90% identity). In contrast, beta 8 is distinct from the other integrin beta subunits. At the amino acid level human beta 8 ranges from 31 to 37% identity with human beta 1-7. The domain structure of beta 8 is typical of the integrin beta subunits. Human beta 8 has a 42-residue N-terminal signal peptide, a large extracellular domain (approximately 639 residues) that contains four cysteine-rich repeats, a transmembrane domain (approximately 30 residues), and a C-terminal cytoplasmic domain (approximately 58 residues). There are several structural features that are unique to the beta 8 polypeptide, as compared with the other integrin beta subunits. Six of the 56 cysteine residues that are conserved within the extracellular domains of beta 1, beta 2, beta 3, beta 5, beta 6, and the beta subunit from Drosophila are absent in the beta 8 polypeptide. Also, the cytoplasmic domain of the beta 8 subunit shares no homology with the cytoplasmic regions of any of the other integrin beta subunits. Northern analysis demonstrated an approximately 8-kilobase beta 8 mRNA in rabbit placenta, kidney, brain, ovary, and uterus. PCR analysis revealed that beta 8 mRNA is also present in several transformed human cell lines. The beta 8 polypeptide has been transiently expressed in 293 human embryonic kidney cells. A polyclonal antipeptide antibody specific for beta 8 and a polyclonal antibody that recognizes alpha v epitopes were used to show that beta 8 can complex with the endogenous alpha v subunit in 293 cells and that the resulting integrin is expressed as a cell surface complex.     

Urokinase-type plasminogen activator receptor induces conformational changes in the integrin alphaMbeta2 headpiece and reorientation of its transmembrane domains

The glycosylphosphatidylinositol-linked urokinase-type plasminogen activator receptor (uPAR) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. Devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules, integrin alpha(5)beta(1), and elicits cytoplasmic signaling. The separation or reorientation of integrin transmembrane domains and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In this investigation we used reporter monoclonal antibodies and fluorescence resonance energy transfer analyses to show conformational changes in the alpha(M)beta(2) headpiece and reorientation of its transmembrane domains when alpha(M)beta(2) interacts with uPAR.     

Structure of the integrin alphaIIb transmembrane segment

Integrin cell-adhesion receptors transduce signals bidirectionally across the plasma membrane via the single-pass transmembrane segments of each alpha and beta subunit. While the beta3 transmembrane segment consists of a linear 29-residue alpha-helix, the structure of the alphaIIb transmembrane segment reveals a linear 24-residue alpha-helix (Ile-966 -Lys-989) followed by a backbone reversal that packs Phe-992-Phe-993 against the transmembrane helix. The length of the alphaIIb transmembrane helix implies the absence of a significant transmembrane helix tilt in contrast to its partnering beta3 subunit. Sequence alignment shows Gly-991-Phe-993 to be fully conserved among all 18 human integrin alpha subunits, suggesting that their unusual structural motif is prototypical for integrin alpha subunits. The alphaIIb transmembrane structure demonstrates a level of complexity within the membrane that is beyond simple transmembrane helices and forms the structural basis for assessing the extent of structural and topological rearrangements upon alphaIIb-beta3 association, i.e. integrin transmembrane signaling.     

Integrin alpha4beta1-dependent adhesion to ADAM 28 (MDC-L) requires an extended surface of the disintegrin domain

ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, alpha4beta1. To elucidate which residues comprise the alpha4beta1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to alpha4beta1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.     

Integrin cytoplasmic domains mediate inside-out signal transduction

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.     

Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells

Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken beta 1 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin beta 1 subunit. The chicken beta 1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD alpha subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD beta 1 and an 140-kD alpha subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse alpha subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.     

Role of the beta-subunit arginine/lysine finger in integrin heterodimer formation and function

Formation of the integrin alphabeta heterodimer is essential for cell surface expression and function. At the core of the alphabeta interface is a conserved Arg/Lys "finger" from the beta-subunit that inserts into a cup-like "cage" formed of two layers of aromatic residues in the alpha-subunit. We evaluated the role of this residue in heterodimer formation in an alphaA-lacking and an alphaA-containing integrin alphaVbeta3 and alphaMbeta2 (CD11b/CD18), respectively. Arg261 of beta3 was mutated to Ala or Glu; the corresponding Lys252 of beta2 was mutated to Ala, Arg, Glu, Asp, or Phe; and the effects on heterodimer formation in each integrin examined by ELISA and immunoprecipitation in HEK 293 cells cotransfected with plasmids encoding the alpha- and beta-subunits. The Arg261Glu (but not Arg261Ala) substitution significantly impaired cell surface expression and heterodimer formation of alphaVbeta3. Although Lys252Arg, and to a lesser extent Lys252Ala, were well tolerated, each of the remaining substitutions markedly reduced cell surface expression and heterodimer formation of CD11b/CD18. Lys252Arg and Lys252Ala integrin heterodimers displayed a significant increase in binding to the physiologic ligand iC3b. These data demonstrate an important role of the Arg/Lys finger in formation of a stable integrin heterodimer, and suggest that subtle changes at this residue affect the activation state of the integrin.     

Collagen XVI harbors an integrin alpha1 beta1 recognition site in its C-terminal domains

Collagen XVI is integrated tissue-dependently into distinct fibrillar aggregates, such as D-banded cartilage fibrils and fibrillin-1-containing microfibrils. In skin, the distribution of collagen XVI overlaps that of the collagen-binding integrins alpha1 beta1 and alpha2 beta1. Basal layer keratinocytes express integrin alpha2 beta1, whereas integrin alpha1 beta1 occurs in smooth muscle cells surrounding blood vessels, in hair follicles, and on adipocytes. Cells bearing the integrins alpha1 beta1 and alpha2 beta1 attach and spread on recombinant collagen XVI. Furthermore, collagen XVI induces the recruitment of these integrins into focal adhesion plaques, a principal step in integrin signaling. Of potential physiological relevance, these integrin-collagen XVI interactions may connect cells with specialized fibrils, thus contributing to the organization of fibrillar and cellular components within connective tissues. In cell-free binding assays, collagen XVI is more avidly bound by alpha1 beta1 integrin than by alpha2 beta1 integrin. Both integrins interact with collagen XVI via the A domain of their alpha subunits. A tryptic collagen XVI fragment comprising the collagenous domains 1-3 is recognized by alpha1 beta1 integrin. Electron microscopy of complexes of alpha1 beta1 integrin with this tryptic collagen XVI fragment or with full-length collagen XVI revealed a unique alpha1 beta1 integrin-binding site within collagen XVI located close to its C-terminal end.     

Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha(M)I-domain of integrin alpha(M)beta2

The leukocyte integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha(M)beta(2) mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma(383)TMKIIPFNRLTIG(395), P2-C, within the gamma-module of the D-domain of fibrinogen, is a recognition site for alpha(M)beta(2) and alpha(X)beta(2). We have now identified the complementary sequences within the alpha(M)I-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha(M)beta(2), alpha(X)beta(2), and alpha(L)beta(2), i.e. the alpha(M)I- and alpha(X)I-domains bind P2-C, and the alpha(L)I-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, which forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimensional structure of the alpha(M)I-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha(M)I-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha(L)I-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr(252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys(245)-Arg(261) segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha(M)I-domains; and 4) grafting the alpha(M)(Lys(245)-Arg(261)) segment into the alpha(L)I-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha(M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha(M)I-, alpha(X)I-, and alpha(L)I-domains, is responsible for recognition of a small segment of fibrinogen, gammaThr(383)-Gly(395), by serving as ligand binding site.     

Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways

L-type, voltage-gated Ca2+ channels (CaL) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle CaL channel isoforms are acutely potentiated following alpha5beta1 integrin activation. Only the alpha1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by alpha5beta1 integrin requires phosphorylation of alpha1C C-terminal residues Ser1901 and Tyr2122. These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following alpha5beta1 integrin activation, native CaL channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of CaL channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.     

Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1)

Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).     

A 50-A separation of the integrin alpha v beta 3 extracellular domain C termini reveals an intermediate activation state

The integrin alpha(v)beta(3) has been shown to exist in low and high affinity conformations. Activation to the high affinity state is thought to depend on the "switchblade-like" opening, from a low affinity bent conformation with a closed headpiece to an extended form of the integrin with an open headpiece. Activation has been shown to depend on separation of the cytoplasmic domains. How cytoplasmic domain separation is related to separation of the transmembrane domains is unknown, and the distance of separation of the transmembrane domains required for activation has not been defined. A constrained secreted form of alpha(v)beta(3) was engineered that introduced a 50-A separation of the integrin C-terminal tails of the extracellular domains of the alpha(v) and beta(3) subunits. Receptor binding and recognition by ligand-induced binding state (LIBS) monoclonal antibodies demonstrated that the mutant receptor was locked into a low affinity state that was likely in a partially extended conformation but with a closed headpiece. In the presence of RGD peptide, the constrained receptor was able to fully extend, as determined by full exposure of LIBS epitopes. In the presence of the appropriate LIBS antibody, high affinity ligand binding of the constrained receptor was achieved. The results support the existence of transient intermediate activation states of secreted alpha(v)beta(3). Furthermore, these results with the secreted alpha(v)beta(3) receptor support a model for the full-length membrane-bound form of alpha(v)beta(3), whereby a 50-A lateral separation of the integrin alpha(v) and beta(3) transmembrane domains would be sufficient to enforce the switchblade-like opening to the extended conformation but insufficient for full receptor activation.     

Involvement of transmembrane domain interactions in signal transduction by alpha/beta integrins

The alpha and beta subunits of alpha/beta heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes. A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data. We have analyzed the capacity of the integrin alpha(2), alpha(IIb), alpha(4), beta(1), beta(3), and beta(7) transmembrane domains to form homodimers and/or heterodimers. Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of alpha and beta subunits; rather, the alpha/beta subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association.     

On the interaction of ribosomal protein L5 with 5S rRNA

Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel beta-sheet and four alpha-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692-701, 20011. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel beta-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at beta-strands and loop structures in BstL5. The mutation of Lys33 at the beta 1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the beta2-strand, the alpha3-beta4 loop, and the beta4-beta5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the beta2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the beta-sheet structure, and Phe77 at the beta2-beta3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the beta-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.     

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.     

The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins

Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed alpha, beta, and gamma. The putative binding site for integrins has been mapped to the G domain of the alpha chain, although trimerization with beta and gamma chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of beta and gamma chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the gamma chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the gamma1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the gamma1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the gamma2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either gamma1 or gamma2 chains. However, the peptide segment modeled after the C-terminal region of gamma1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.