Distinct Conformational Changes Research Articles

Structural Dynamics of α-Actinin-Vinculin Interactions

Alpha-actinin and vinculin orchestrate reorganization of the actin cytoskeleton following the formation of adhesion junctions. alpha-Actinin interacts with vinculin through the binding of an alpha-helix (alphaVBS) present within the R4 spectrin repeat of its central rod domain to vinculin's N-terminal seven-helical bundle domain (Vh1). The Vh1:alphaVBS structure suggests that alphaVBS first unravels from its buried location in the triple-helical R4 repeat to allow it to bind to vinculin. alphaVBS binding then induces novel conformational changes in the N-terminal helical bundle of Vh1, which disrupt its intramolecular association with vinculin's tail domain and which differ from the alterations in Vh1 provoked by the binding of talin. Surprisingly, alphaVBS binds to Vh1 in an inverted orientation compared to the binding of talin's VBSs to vinculin. Importantly, the binding of alphaVBS and talin's VBSs to vinculin's Vh1 domain appear to also trigger distinct conformational changes in full-length vinculin, opening up distant regions that are buried in the inactive molecule. The data suggest a model where vinculin's Vh1 domain acts as a molecular switch that undergoes distinct structural changes provoked by talin and alpha-actinin binding in focal adhesions versus adherens junctions, respectively.

Mg2+ and Ca2+ Differentially Regulate DNA Binding and Dimerization of DREAM

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca(2+) binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca(2+) binding to the various mutants revealed that, in the absence of Mg(2+), Ca(2+) binds independently and sequentially to EF-3 (DeltaH = -2.4 kcal/mol), EF-4 (DeltaH = +5.2 kcal/mol), and EF-2 (DeltaH = +1 kcal/mol). By contrast, only two Ca(2+) bind to DREAM in the presence of physiological levels of Mg(2+) for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg(2+). ITC measurements demonstrate that one Mg(2+) binds enthalpically with high affinity (K(d) = 13 mum and DeltaH = -0.79 kcal/mol) and two or more Mg(2+) bind entropically in the millimolar range. Size-exclusion chromatography studies revealed that Mg(2+) stabilizes DREAM as a monomer, whereas Ca(2+) induces protein dimerization. Electrophoretic mobility shift assays indicated that Mg(2+) is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca(2+) levels, suggesting that binding of a single Ca(2+) at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca(2+) and Mg(2+) induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg(2+) binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca(2+)-induced protein dimerization disrupts DNA binding.

Interaction of self-assembled monolayers of oligo(ethylene glycol)-terminated alkanethiols with water studied by vibrational sum-frequency generation

Vibrational sum-frequency generation (VSFG) was used to investigate the conformational changes in self-assembled monolayers (SAMs) of (1-mercaptoundec-11-yl) hexa(ethylene glycol) monomethylether (EG6-OMe) on gold when exposed to liquid water. VSFG spectra of the EG6-OMe SAMs were recorded before, during, and after exposure of the films to water and after a subsequent evacuation step. While in contact with water the entire ethylene glycol chains are found in a random, solvated state, after removal from the fluid water molecules remain absorbed only at the terminal groups of the film giving rise to distinct conformational changes. After evacuation, the structure of the EG6-OMe SAM reverts to its original state, indicating that water has been removed from the monolayer. Our findings support recent ab initio calculations and Monte Carlo simulations on the interaction of ethylene glycol-terminated monolayers with water.

Kinetic Analysis and Ligand-Induced Conformational Changes in Dimeric and Tetrameric Forms of Human Thymidine Kinase 2

Recombinant human thymidine kinase 2 (hTK2) expressed in Escherichia coli has been found to bind tightly a substoichiometric amount of deoxyribonucleoside triphosphates (dTTP > dCTP >> dATP), known to be strong feedback inhibitors of the enzyme. Incubation of hTK2 with the substrate dThd was able to release the dNTPs from the active site during purification from E. coli and thus allowed the kinetic characterization of the noninhibited enzyme, with the tetrameric hTK2 showing slightly higher activity than the most abundant dimeric form. The unliganded hTK2 revealed a lower structural stability than the inhibitor-bound enzyme forms, being more prone to aggregation, thermal denaturation, and limited proteolysis. Moreover, intrinsic tryptophan fluorescence (ITF), far-UV circular dichroism (CD), and limited proteolysis have revealed that hTK2 undergoes distinct conformational changes upon binding different substrates and inhibitors, which are known to occur in the nucleoside monophosphate kinase family. The CD-monitored thermal denaturation of hTK2 dimer/tetramer revealed an irreversible process that can be satisfactorily described by the two-state irreversible denaturation model. On the basis of this model, the parameters of the Arrhenius equation were calculated, providing evidence for a significant structural stabilization of the enzyme upon ligand binding (dCyd < MgdCTP < dThd < dCTP < dTTP < MgdTTP), whereas MgATP further destabilizes the enzyme. Finally, surface plasmon resonance (SPR) was used to study in real time the reversible binding of substrates and inhibitors to the immobilized enzyme. The binding affinities for the inhibitors were found to be 1-2 orders of magnitude higher than for the corresponding substrates, both by SPR and ITF analysis.

Improved Lymphocyte Function-associated Antigen-1 (LFA-1) Inhibition by Statin Derivatives: MOLECULAR BASIS DETERMINED BY X-RAY ANALYSIS AND MONITORING OF LFA-1 CONFORMATIONAL CHANGES IN VITRO AND EX VIVO

The integrin lymphocyte function-associated antigen-1 (LFA-1) (alphaLbeta2; CD11a/CD18) plays an important role in leukocyte migration and T cell activation. LFA-1 is inhibited by the cholesterol-lowering drug lovastatin, which binds to an allosteric site of the alphaL I domain termed the lovastatin site (L-site). Here we report for the first time the x-ray structures of the LFA-1 I domain complexed with derivatives of lovastatin optimized for LFA-1 inhibition. This analysis identified two new subpockets within the L-site occupied by chemical groups of the statin derivatives but not by lovastatin itself. Occupancy of these L-site subpockets led to distinct conformational changes in LFA-1, which were detectable by an epitope-monitoring assay. We utilized this assay to demonstrate improved LFA-1 inhibition in human blood in vitro and in blood samples from treated animals ex vivo. Moreover, we demonstrate that the novel lovastatin-derived LFA-1 inhibitor LFA878 exhibits potent anti-inflammatory effects in carrageenan-induced rat paw edema. In summary, the findings reported here extend the understanding of LFA-1 inhibition at the molecular level, allow for the identification and design of LFA-1 inhibitors of further enhanced potency, and support the expectation that LFA-1 inhibitors binding to the L-site will be of therapeutic value in treating inflammatory diseases.

Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis.

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K(d) values of 7.9, 6.9, and 0.28 microM, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K(d) values of 1.1 and 0.73 microM, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V(max)/K(m)) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.

Mutations in the nucleotide-binding domain of MutS homologs uncouple cell death from cell survival

After genotoxic insult, the decision to repair or undergo cell death is pivotal for undamaged cell survival, and requires a highly controlled coordination of both pathways. Disruption of this regulation results in tumorigenesis and failure of cancer therapy. Mismatch repair (MMR) proteins have a unique role by contributing to both pathways, though direct evidence for their function in the DNA damage response is ambiguous. We report separation of function mutants in the ATPase domains of yeast MutS homologous (MSH) proteins that uncouple MMR-dependent DNA repair from damage response to cisplatin. While mutations in the ATPase domain have devastating effects on the mutation rate of the cell, ATPase processing is mostly dispensable for the cell death phenotype; only limited processing by the MSH6 subunit is required in DNA damage response. Different DNA binding patterns and nucleotide sensitivity of Msh2/Msh6–DNA adduct and protein-mismatch complexes, respectively, suggest that the presence of different DNA lesions influences the requirement for ATP. Limited proteolysis of purified protein gives first indications for differences in nucleotide-induced conformational changes in the presence of platinated DNA. Structural modeling of bacterial MutS proteins reinforces nucleotide-dependent differences in structures that contribute to the distinction between DNA damage response and repair. Our results demonstrate the uncoupling of MMR-dependent damage response from repair and present first indications for the involvement of distinct conformational changes in MSH proteins in this process. These data present evidence for a mechanism of MMR-dependent damage response that differs from MMR; these results have strong implications for the chemotherapeutic treatment of MMR-defective tumors.

Distortion of the Catalytic Domain of Tissue-type Plasminogen Activator by Plasminogen Activator Inhibitor-1 Coincides with the Formation of Stable Serpin-Proteinase Complexes

Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.

Structure of the Oxygen Sensor in Bacillus subtilis: Signal Transduction of Chemotaxis by Control of Symmetry

Much is now known about chemotaxis signaling transduction for Escherichia coli and Salmonella typhimurium. The mechanism of chemotaxis of Bacillus subtilis is, in a sense, reversed. Attractant binding strengthens the activity of histidine kinase in B. subtilis, instead of an inhibition reaction. The HemAT from B. subtilis can detect oxygen and transmit the signal to regulatory proteins that control the direction of flagella rotation. We have determined the crystal structures of the HemAT sensor domain in liganded and unliganded forms at 2.15 Å and 2.7 Å resolution, respectively. The liganded structure reveals a highly symmetrical organization. Tyrosine70 shows distinct conformational changes on one subunit when ligands are removed. Our study suggests that disruption of the symmetry of HemAT plays an important role in initiating the chemotaxis signaling transduction cascade.

Functional analysis of genetic mutations in nucleotide binding domain 2 of the human retina specific ABC transporter.

The rod outer segment (ROS) ABC transporter (ABCR) plays an important role in the outer segment of retinal rod cells, where it functions as a transporter of all-trans retinal, most probably as the complex lipid, retinylidene-phosphatidyl-ethanolamine. We report here a quantitative analysis of the structural and functional effects of genetic mutations, associated with several macular degenerations, in the second nucleotide-binding domain of ABCR (NBD2). We have analyzed the ATP binding, kinetics of ATP hydrolysis, and structural changes. The results of these multifaceted analyses were correlated with the disease severity and prognosis. Results presented here demonstrated that, in wild type NBD2, distinct conformational changes accompany nucleotide (ATP and ADP) binding. Upon ATP binding, NBD2 protein changed to a relaxed conformation where tryptophans became more solvent-exposed, while ADP binding reverses this process and leads back to a taut conformation that is also observed with the unbound protein. This sequence of conformational change appears to be important in the energetics of the ATP hydrolysis and may have important structural consequences in the ability of the NBD2 domain to act as a regulator of the nucleotide-binding domain 1. Some of the mutant proteins displayed strikingly different patterns of conformational changes upon nucleotide binding that pointed to unique structural consequences of these genetic mutations. The ABCR dysfunctions, associated with various retinopathies, are multifaceted in nature and include alterations in protein structure as well as the attenuation of ATPase activity and nucleotide binding.

Substrate-induced conformational changes in Escherichia coli arginyl–tRNA synthetase observed by 19F NMR spectroscopy

The 19F nuclear magnetic resonance (NMR) spectra of 4-fluorotryptophan (4-F-Trp)-labeled Escherichia coli arginyl–tRNA synthetase (ArgRS) show that there are distinct conformational changes in the catalytic core and tRNA anticodon stem and loop-binding domain of the enzyme, when arginine and tRNA Arg are added to the unliganded enzyme. We have assigned five fluorine resonances of 4-F-Trp residues (162, 172, 228, 349 and 446) in the spectrum of the fluorinated enzyme by site-directed mutagenesis. The local conformational changes of E. coli ArgRS induced by its substrates observed herein by 19F NMR are similar to those of crystalline yeast homologous enzyme.

Von Willebrand factor

The adhesive protein von Willebrand factor contributes to platelet function by mediating the initiation and progression of thrombus formation at sites of vascular injury. In the last 2 years, there has been considerable progress in explaining the biologic properties of von Willebrand factor. The three-dimensional structure of specific domains has been explained, with the demonstration of distinct conformational changes in the A1 domain caused by single amino acid substitutions associated with enhanced binding to platelets. The structural and functional properties of the interaction between the von Willebrand factor A1 domain and glycoprotein Ibalpha have also been elucidated in greater detail, bringing researchers closer to understanding how this adhesive bond can oppose the fluid dynamic effects of rapidly flowing blood to initiate thrombus formation and, concurrently, contribute to platelet activation. Because hemodynamic forces greatly influence platelet responses to vascular injury in stenosed and partially occluded arteries, a detailed description of how von Willebrand factor interacts with tissues and platelets may help in the design of more specific therapeutic inhibitors of arterial thrombosis. Moreover, enlightening findings have been obtained on the link between regulation of von Willebrand factor multimer size and microvascular thrombosis. This progress in basic research has provided critical information to define with greater precision the role of von Willebrand factor in vascular biology and pathology.

Von Willebrand factor.

The adhesive protein von Willebrand factor contributes to platelet function by mediating the initiation and progression of thrombus formation at sites of vascular injury. In the last 2 years, there has been considerable progress in explaining the biologic properties of von Willebrand factor. The three-dimensional structure of specific domains has been explained, with the demonstration of distinct conformational changes in the A1 domain caused by single amino acid substitutions associated with enhanced binding to platelets. The structural and functional properties of the interaction between the von Willebrand factor A1 domain and glycoprotein Ibalpha have also been elucidated in greater detail, bringing researchers closer to understanding how this adhesive bond can oppose the fluid dynamic effects of rapidly flowing blood to initiate thrombus formation and, concurrently, contribute to platelet activation. Because hemodynamic forces greatly influence platelet responses to vascular injury in stenosed and partially occluded arteries, a detailed description of how von Willebrand factor interacts with tissues and platelets may help in the design of more specific therapeutic inhibitors of arterial thrombosis. Moreover, enlightening findings have been obtained on the link between regulation of von Willebrand factor multimer size and microvascular thrombosis. This progress in basic research has provided critical information to define with greater precision the role of von Willebrand factor in vascular biology and pathology.

Activation of NR1/NR2B NMDA receptors.

N-methyl-D-aspartate (NMDA) receptors are highly expressed in the central nervous system and are involved in excitatory synaptic transmission as well as synaptic plasticity. Despite considerable structural and biophysical research, the mechanism behind activation of the NMDA receptor is still poorly understood. By analyzing patch clamp recordings of one channel activated by glutamate, we determined the burst structure and open probability for recombinant rat NR1/NR2B receptors. We used partial agonists at the glutamate and glycine binding sites to show that at least two kinetically distinct subunit-associated conformational changes link co-agonist binding to the opening of the NMDA receptor pore. These data suggest that NR1 and NR2B subunits, respectively, undergo a fast and slow agonist-dependent conformational change that precedes opening of the pore. We propose a new working model of receptor activation that can account for macroscopic as well as microscopic NMDA receptor properties.

Capillary electrophoretic study on pH dependence of enantioselective disopyramide binding to genetic variants of human alpha1-acid glycoprotein.

A high-performance frontal analysis-capillary electrophoresis (HPFA-CE) method was applied to investigate the effect of pH on the drug binding properties of genetic variants of human alpha1-acid glycoprotein (AGP), A variant and a mixture of F1S variants. The unbound concentrations of a model basic drug, disopyramide (DP), in A variant solutions and in F1S variant solutions were measured by HPFA-CE to evaluate binding constants at pH 4.0, 5.0, 6.0 and 7.4. The binding between DP and A variant was gradually weakened by acidification of background buffer (from pH 7.4 to 4.0), while the binding between DP and FIS variants decreased at first (from pH 7.4 to 6.0), and then gained (from pH 6.0 to 4.0). Consequently, DP was more strongly bound to A variant than to FIS variants at pH 7.4, while at pH 4.0 DP was more strongly bound to F1S variants. At any pH (S)-DP was bound more strongly than (R)-DP, and the enantioselectivity of A variant was significantly higher than that of F1S variants. Electrophoretic mobilities of the AGP genetic variants decreased along with a decrease in pH. Fluorescent emission of these genetic variants indicated a distinct conformational change between pH 5.0 and 4.0. However, there was no significant difference in the electrophoretic mobility and the fluorescent emission spectrum between these variants at any pH. On the other hand, circular dichroism analyses revealed that beta-sheet content in FIS variants diminished as pH decreased, while that in A variant increased. These results suggest that the conformational change induced by acidification of background buffer differs between these genetic variants, and this causes the difference in DP bindability.

The RNA Helicase DbpA Exhibits a Markedly Different Conformation in the ADP-bound State When Compared with the ATP- or RNA-Bound States

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.

Species-specific pheromonal compounds induce distinct conformational changes of pheromone binding protein subtypes from Antheraea polyphemus.

We have investigated the structural features of three pheromone binding protein (PBP) subtypes from Antheraea polyphemus and monitored possible changes induced upon interaction with the Antheraea pheromonal compounds 4E,9Z-14:Ac [(E4,Z9)-tetradecadienyl-1-acetate], 6E,11Z-16:Ac [(E6,Z11)-hexadecadienyl-1-acetate], and 6E,11Z-16:Al [(E6,Z11)-hexadecadienal]. Circular dichroism and second derivative UV-difference spectroscopy data demonstrate that the structure of subtype PBP1 significantly changes upon binding of 4E,9Z-14:Ac. The related 6E,11Z-16:Ac was less effective and 6E,11Z-16:Al showed only a small effect. In contrast, in subtype PBP2 pronounced structural changes were only induced by the 6E,11Z-16:Al, and the subtype PBP3 did not show any considerable changes in response to the pheromonal compounds. The UV-spectroscopic data suggest that histidine residues are likely to be involved in the ligand-induced structural changes of the proteins, and this notion was confirmed by site-directed mutagenesis experiments. These results demonstrate that appropriate ligands induce structural changes in PBPs and provide evidence for ligand specificity of these proteins.

Mutations that bypass tRNA binding activate the intrinsically defective kinase domain in GCN2.

The protein kinase GCN2 is activated in amino acid-starved cells on binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-related domain. We isolated two point mutations in the protein kinase (PK) domain, R794G and F842L, that permit strong kinase activity in the absence of tRNA binding. These mutations also bypass the requirement for ribosome binding, dimerization, and association with the GCN1/GCN20 regulatory complex, suggesting that all of these functions facilitate tRNA binding to wild-type GCN2. While the isolated wild-type PK domain was completely inert, the mutant PK was highly active in vivo and in vitro. These results identify an inhibitory structure intrinsic to the PK domain that must be overcome on tRNA binding by interactions with a regulatory region, most likely the N terminus of the HisRS segment. As Arg 794 and Phe 842 are predicted to lie close to one another and to the active site, they may participate directly in misaligning active site residues. Autophosphorylation of the activation loop was stimulated by R794G and F842L, and the autophosphorylation sites remained critical for GCN2 function in the presence of these mutations. Our results imply a two-step activation mechanism involving distinct conformational changes in the PK domain.

Integrin Signals Specified from Without

Integrins are receptors that interact with the extracellular matix and transmit signals into the cytoplasm. Integrin signals lead to activation of the small guanosine triphosphatases Cdc42, Rac, and Rho. Miao et al. used overexpression of integrin β3 subunits in Chinese hamster ovary cells to show that β3 integrin is coupled to activation of Rho and formation of stress fibers, whereas activation of β1 integrin is coupled to activation of Rac and formation of lamellipodia. The specificity of ligand binding of β integrin subunits is determined by an extracellular region known as the I domain. Replacement of the I domain of β1 integrin with the I domain from β3 integrin caused the resulting chimeric integrin to have ligand-binding specificity of β3 integrin and to couple to activation of Rho rather than Rac. The results indicate that β1 and β3 integrins are coupled to distinct signaling outputs. And, it appears that, depending on which extracellular I domain is present, ligand binding may cause distinct conformational changes in the β subunit that lead to coupling of the intracellular portion of the receptor to alternate signaling partners.H. Miao, S. Li, Y.-L. Hu, S. Yuan, Y. Zhao, B. P. C. Chen, W. Puzon-McLaughlin, T. Tarui, J. Y.-J. Shyy, Y. Takada, S. Usami, S. Chien, Differential regulation of Rho GTPases by β1 and β3 integrins: The role of an extracellular domain of integrin in intracellular signaling. J. Cell Sci. 115, 2199-2206 (2002). [Online Journal]

Integrin Signals Specified from Without

Integrins are receptors that interact with the extracellular matix and transmit signals into the cytoplasm. Integrin signals lead to activation of the small guanosine triphosphatases Cdc42, Rac, and Rho. Miao et al. used overexpression of integrin β3 subunits in Chinese hamster ovary cells to show that β3 integrin is coupled to activation of Rho and formation of stress fibers, whereas activation of β1 integrin is coupled to activation of Rac and formation of lamellipodia. The specificity of ligand binding of β integrin subunits is determined by an extracellular region known as the I domain. Replacement of the I domain of β1 integrin with the I domain from β3 integrin caused the resulting chimeric integrin to have ligand-binding specificity of β3 integrin and to couple to activation of Rho rather than Rac. The results indicate that β1 and β3 integrins are coupled to distinct signaling outputs. And, it appears that, depending on which extracellular I domain is present, ligand binding may cause distinct conformational changes in the β subunit that lead to coupling of the intracellular portion of the receptor to alternate signaling partners. H. Miao, S. Li, Y.-L. Hu, S. Yuan, Y. Zhao, B. P. C. Chen, W. Puzon-McLaughlin, T. Tarui, J. Y.-J. Shyy, Y. Takada, S. Usami, S. Chien, Differential regulation of Rho GTPases by β1 and β3 integrins: The role of an extracellular domain of integrin in intracellular signaling. J. Cell Sci. 115 , 2199-2206 (2002). [Online Journal]