Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface.In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

TGF-beta1 enhances betaig-h3-mediated keratinocyte cell migration through the alpha3beta1 integrin and PI3K

betaig-h3 is an extracellular matrix (ECM) protein whose expression is highly induced by transforming growth factor beta1 (TGF-beta1). We previously demonstrated that betaig-h3 has two alpha3beta1 integrin-interacting motifs, which promote adhesion, migration, and proliferation of human keratinocytes. Both betaig-h3 and TGF-beta1 have been suggested to play important roles in the healing of skin wounds. In this study, we demonstrate that TGF-beta1 enhances keratinocyte adhesion and migration toward betaig-h3 through the alpha3beta1 integrin. TGF-beta1 did not increase the amount of the alpha3beta1 integrin on the cell surface, but rather increased its affinity for betaig-h3. LY294002, an inhibitor of PI3K, blocked the basal and TGF-beta1-enhanced cell migration but not adhesion to betaig-h3. A constitutively active mutant of PI3K stimulated cell migration but not adhesion to betaig-h3. The PI3K pathway is also not associated with the affinity of the alpha3beta1 integrin to betaig-h3. TGF-beta1 induced phosphorylation of AKT and FAK. Taken together, these data suggest that TGF-beta1 increases affinity of the alpha3beta1 integrin to betaig-h3, resulting in enhanced adhesion and migration of keratinocytes toward betaig-h3. TGF-beta1 also enhances migration through PI3K, but PI3K is not associated with either the binding affinity of the alpha3beta1 integrin or its adhesion to betaig-h3.     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.     

Spatial restriction of alpha4 integrin phosphorylation regulates lamellipodial stability and alpha4beta1-dependent cell migration

Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.     

EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.

We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin alpha6beta4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the beta4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with alpha6beta4. By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.     

Autocrine laminin-5 ligates alpha6beta4 integrin and activates RAC and NFkappaB to mediate anchorage-independent survival of mammary tumors

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express alpha6beta4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail-truncated alpha6beta4 integrin. alpha6beta4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of beta4 integrin is necessary for basal and epidermal growth factor-induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional beta1 and beta4 integrin through activation of NFkappaB, and overexpression of NFkappaB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5-alpha6beta4 integrin-RAC-NFkappaB signaling.     

Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin

The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.     

Expression of Integrin ��v��6 and TGF-�� in Scarless vs Scar-forming Wound Healing

Oral mucosal wounds heal with reduced scar formation compared with skin. The epithelial integrin αvβ6 is induced during wound healing, and it can activate fibrogenic transforming growth factor β1 (TGF-β1) and anti-fibrogenic TGF-β3 that play key roles in scar formation. In this study, expression of β6 integrin and members of the TGF-β pathway were studied in experimental wounds of human gingiva and both gingiva and skin of red Duroc pigs using real-time PCR, gene microarrays, and immunostaining. Similar to human wounds, the expression of β6 integrin was induced in the pig wounds 7 days after wounding and remained upregulated >49 days. The αvβ6 integrin was colocalized with both TGF-β isoforms in the wound epithelium. Significantly higher expression levels of β6 integrin and TGF-β1 were observed in the pig gingival wounds compared with skin. Early gingival wounds also expressed higher levels of TGF-β3 compared with skin. The spatio-temporal colocalization of αvβ6 integrin with TGF-β1 and TGF-β3 in the wound epithelium suggests that αvβ6 integrin may activate both isoforms during wound healing. Prolonged expression of αvβ6 integrin along with TGF-β3 in the gingival wound epithelium may be important in protection of gingiva from scar formation. (J Histochem Cytochem 57:543–557, 2009)     

Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active

Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. In the present study, we identified clusters of integrin that formed upstream of Rac activation. These clusters contained a Rac-binding protein(s) and appeared to be involved in Rac activation. The integrin clusters contained calpain and calpain-cleaved beta3 integrin, while the focal complexes and focal adhesions that formed once Rac and Rho were activated did not. Moreover, the integrin clusters were dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases were dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurred even when calpain inhibitors were present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s).     

Endothelial cell migration on fibronectin is regulated by syntaxin 6-mediated alpha5beta1 integrin recycling

The α5β1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi- and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for α5β1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and α5β1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of β1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated α5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of α5β1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.     

Aza-bicyclic amino acid carboxamides as alpha4beta1/alpha4beta7 integrin receptor antagonists

A series of N-carboxy, N-alkyl, and N-carboxamido azabicyclo[2.2.2]octane carboxamides were prepared and assayed for inhibition of alpha4beta1-VCAM-1 and alpha4beta7-MAdCAM-1 interactions. Potency and alpha4beta1/alpha4beta7 selectivity were sensitive to the substituent R1-R3 in the structures 6, 7, and 8. Several compounds demonstrated low nanomolar balanced alpha4beta1/alpha4beta7 in vitro activity. Two compounds were selected for in vivo leukocytosis studies and demonstrated increases in circulating lymphocytes up to 250% over control.     

Novel Roles for α3β1 Integrin as a Regulator of Cytoskeletal Assembly and as a Trans-dominant Inhibitor of Integrin Receptor Function in Mouse Keratinocytes

Previously we found that α3β1 integrin–deficient neonatal mice develop micro-blisters at the epidermal–dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of α3β1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of α3β1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of α3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and α-actinin at focal contact sites in the α3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to α3β1 deficiency. Apart from the loss of α3β1 there is no change in expression of the other integrins expressed by the α3-null keratinocytes. However, in functional assays, α3β1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of α3β1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.     

Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.

Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits. The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.     

Phactr4: A new integrin modulator required for directional migration of enteric neural crest cells

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.     

RGD targeted poly(L-glutamic acid)-cystamine-(Gd-DO3A) conjugate for detecting angiogenesis biomarker alpha(v) beta3 integrin with MRT, mapping

Cyclic Arg-Gly-Asp-D-Phe-Lys [c(RGDfK)] targeted poly(L-glutamic acid) (PGA)-(Gd-DO3A) conjugate with a biodegradable cystamine spacer was prepared and evaluated for in vivo detection of an angiogenesis biomarker, alpha(v)beta3 integrin, in neoplastic tissues with T1 mapping, a quantitative magnetic resonance imaging (MRI) technique. The binding activity of the c(RGDfK) containing conjugate was investigated using in vitro vitronectin assay with human prostate carcinoma DU145 cell line and Kaposi's sarcoma SLK cell line. The peptide c(RGDfK) and PGA-cystamine-(Gd-DO3A) conjugate were used as controls. The binding affinity of polymer bound c(RGDfK) was slightly lower than free c(RGDfK) peptide. The RGD targeted conjugate had higher binding affinity to the DU145 cells than the SLK cells, which was consistent to free c(RGDfK). The imaging of alpha(v)beta3 integrin with targeted PGA-cystamine-(Gd-DO3A) was evaluated in nude mice bearing DU145 and SLK xenografts at a dose of 5 micromol-Gd/kg. The targeted conjugate demonstrated higher in vivo binding affinity to the DU145 xenografts than the SLK xenografts, resulting in a significant decrease of T1 values of water protons in the periphery of the DU145 tumors as shown in the MR T1 maps. No significant decrease of T1 values was observed in the SLK tumor with the targeted conjugate and in both tumors with the non-targeted conjugate. The targeted polymeric Gd(III) chelate conjugate with a degradable spacer has the potential to be a new paradigm for safe and effective probes in molecular imaging with quantitative MR T1 mapping.