Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces.

A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein-protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds.

The membrane-cytoplasm interface of integrin alpha subunits is critical for receptor latency.

Localization of integrin receptors to focal contact sites occurs upon ligand binding. This activity is latent, since unoccupied integrin receptors do not localize to focal contacts. Deletion analysis has revealed that the alpha cytoplasmic domains is required for the maintenance of integrin receptor latency. Our current hypothesis for the mechanism of integrin post-ligand binding events is that there is a change in relationship of alpha and beta cytoplasmic domains, which overcomes receptor latency. One possible mechanism for such a change would involve the amino acid residues at the membrane-cytoplasm interface. To test this hypothesis, we have produced point mutations in the human integrin alpha 1 subunit. These mutations had no effect on the adhesion via alpha 1 beta 1 to its ligand, collagen IV. However, receptor latency is lost in one of these mutants, leading to constitutive focal contact localization. This effect did not occur in receptors with an exchange of intracellular domains, suggesting that the mechanism of loss of latency involves a relative motion of the integrin chains. These results suggest a model in which post-ligand binding events in integrin receptors are associated with changes in the position of the alpha and beta cytoplasmic domains.

Cell adhesion to extracellular matrix regulates the life cycle of integrins.

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.

Assembly and function of integrin receptors is dependent on opposing alpha and beta cytoplasmic domains.

The membrane proximal regions of integrin alpha and beta subunits are highly conserved in evolution. In particular, all integrin alpha subunits share the KXGFFKR sequence at the beginning of their cytoplasmic domains. Previous work has shown that this domain is important in integrin receptor assembly. Using chimeric integrin alpha and beta subunits, we show that the native cytoplasmic domains of both subunits must be present for efficient assembly. Most strikingly, chimeric alpha 1 and beta 1 subunits with reciprocally swapped intracellular domains dimerize selectively into collagen IV receptors expressed at high levels on the surface. However, these receptors, which bind ligand efficiently, are deficient in a variety of post-ligand binding events, including cytoskeletal association and induction of tyrosine phosphorylation. Furthermore, deletion of the distal alpha cytoplasmic domain in the swapped heterodimers leads to ligand-independent focal contact localization, which also occurs in wild-type subunits when the distal cytoplasmic domain is deleted. These results show that proper integrin assembly requires opposed alpha and beta cytoplasmic domains, and this opposition prevents ligand-independent focal contact localization. Our working hypothesis is that these two domains may associate during receptor assembly and provide the mechanism for integrin receptor latency.

Integrin beta 1 cytoplasmic domain dominant negative effects revealed by lysophosphatidic acid treatment.

Integrin receptors localize to focal contact sites and interact with the cytoskeleton via the beta 1 cytoplasmic domain. To study the role of this domain in adhesion, we have expressed in NIH 3T3 cells a cDNA consisting of the interleukin 2 receptor alpha subunit extracellular and transmembrane domains, connected to the integrin beta 1 cytoplasmic domain (IL2R-beta 1). Since the extracellular domain of the chimeric protein has no role in adhesion, this protein could uncouple adhesion from intracellular events. As expected, in a cell line expressing IL2R-beta 1, this chimera was directed to focal contact sites. Unexpectedly, the cells exhibited normal adhesion to fibronectin (FN). However, when a rapid reorganization of the cytoskeleton was induced using lysophosphatidic acid (LPA), IL2R-beta 1 cells detached from FN in contrast to wild-type cells. The detachment in response to LPA could be prevented with cytochalasin D, an inhibitor of actin polymerization. These results imply that a beta 1 cytoplasmic domain, which is uncoupled from adhesion, can compete with the cytoplasmic domain of native integrin beta 1 for cytoskeletal proteins. As a consequence, the IL2R-beta 1 protein acts as a dominant negative effector of adhesion by disrupting the integrin-cytoskeleton connection.

The role of the I domain in ligand binding of the human integrin alpha 1 beta 1.

We report here the analysis of potential ligand binding domains within the human integrin alpha 1 subunit, a known collagen/laminin receptor. This integrin is effectively blocked by the mouse monoclonal antibody 1B3.1. A truncated version of the alpha 1 subunit lacking the NH2-terminal half of the extracellular domain is not recognized by monoclonal antibody 1B3.1. Furthermore, we have isolated a cDNA containing the I domain from chicken alpha 1 bearing significant homology to the human and rat alpha 1 sequences. Replacing the human I domain with its chicken counterpart led to the surface expression of a functional heterodimer with endogenous mouse beta 1 on NIH 3T3 cells. However, 1B3.1 does not bind to the chicken/human chimera, demonstrating that the human alpha 1 I domain is required for epitope recognition. Mutation of Asp253 within the I domain to alanine resulted in surface expression of an alpha beta heterodimer recognized by 1B3.1 but with markedly reduced binding to collagen IV or laminin. Since a previously reported mutation of a homologous Asp in the Mac-1 I domain has similar consequences, these results suggest a central role for the I domain in ligand recognition for all integrin alpha subunits containing this domain.

Ligand-dependent and -independent integrin focal contact localization: the role of the alpha chain cytoplasmic domain.

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta 1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha 1 wild-type subunit (HA1 cells) or the cytoplasmic domain deleted alpha 1 subunit (CYT cells). Both cell lines express similar levels of the human alpha 1 subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha 1 beta 1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha 5 beta 1 but not for alpha 1 beta 1, there is a striking difference in the cellular localization of alpha 1 beta 1. The HA1 cells show only alpha 5 in focal contacts, without alpha 1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha 1 appears in focal contacts along with the alpha 5/beta 1. Thus, there is both ligand-dependent (alpha 5/beta 1) and ligand-independent (alpha 1/beta 1) focal contact localization in these cells. The truncated alpha 1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha 1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.

Expression of native and truncated forms of the human integrin alpha 1 subunit.

We report here the molecular cloning of cDNAs encoding for the human integrin alpha 1 subunit. The sequence is characteristic of an I domain containing integrin alpha subunit, with a high degree of homology to the rat integrin alpha 1 subunit, including complete identity of the transmembrane and cytoplasmic domains between the two species. The human cDNA directs the expression in mouse NIH 3T3 cells of authentic human alpha 1 protein as demonstrated by the reactivity of this subunit with two human-specific anti-alpha 1 monoclonal antibodies. This exogenous integrin specifically binds to type IV collagen in a Mg(2+)-dependent fashion. We have expressed in both transient systems and in stable cell lines truncated, soluble forms of the human alpha 1 subunit combined with truncated, soluble forms of beta 1 subunits. Although soluble beta 1 subunit was found in the media when the corresponding cDNA was used, the secretion of the soluble alpha 1 subunit was found to be dependent on dimerization with soluble beta 1. Co-transfection of truncated human alpha 1 cDNA with truncated forms of either the human or avian beta 1 cDNA led to efficient secretion of alpha 1.beta 1 heterodimers. These soluble heterodimers specifically bind to collagen IV in a manner similar to their full-length counterparts. Biosynthetic studies using stably expressing cell lines demonstrate that the soluble heterodimers and the native heterodimers are formed independently, strongly suggesting that the transmembrane or cytoplasmic domains of alpha and beta subunits are involved in the assembly of native heterodimers.

DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance.

Staphylococcus aureus gyrA and gyrB genes, which encode the DNA gyrase A and B proteins, have been isolated and found to map contiguously. DNA sequence analysis revealed close homology between the S. aureus gyrase subunits and their counterparts in Bacillus subtilis and Escherichia coli, including several conserved amino acid residues whose substitution in E. coli confers resistance to 4-quinolones. These results are discussed in regard to quinolone resistance mechanisms in S. aureus.