We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.

The ␣9␤1 integrin accelerates cell migration through binding of spermidine/spermine acetyltransferase (SSAT) to the ␣9 cytoplasmic domain. We now show that SSAT enhances ␣9-mediated migration specifically through catabolism of spermidine and/or spermine. Because spermine and spermidine are effective blockers of K ؉ ion efflux through inward-rectifier K ؉ (Kir) channels, we examined the involvement of Kir channels in this pathway. The Kir channel inhibitor, barium, or knockdown of a single subunit, Kir4.2, specifically inhibited ␣9-dependent cell migration. ␣9␤1 and Kir4.2 colocalized in focal adhesions at the leading edge of migrating cells and inhibition or knockdown of Kir4.2 caused reduced persistence and an increased number of lamellipodial extensions in cells migrating on an ␣9␤1 ligand. These results identify a pathway through which the ␣9 integrin subunit stimulates cell migration by localized polyamine catabolism and modulation of Kir channel function.T he ␣9␤1 integrin is widely expressed and binds to a number of ligands, including the extracellular matrix protein tenascin-C, the vascular cell adhesion molecule, VCAM-1, and several members of the ADAM family (1-3). ␣9␤1 mediates enhanced cell migration, an effect that specifically depends on the ␣9 cytoplasmic domain (4, 5). The closely related integrin ␣4 subunit also accelerates cell migration, through reversible interaction with the scaffolding protein, paxillin (6, 7). The mechanism by which the ␣9 subunit cytoplasmic domain accelerates cell migration is completely different (4, 5), and requires binding of the enzyme, spermidine/ spermine-N 1 -acetyltransferase (SSAT) (5). The mechanisms by which SSAT binding modulates migration were until now unknown. SSAT specifically catalyzes catabolism of the higher order polyamines, spermidine and spermine, to the lower order polyamine, putrescine (8), thereby increasing intracellular levels of putrescine, and decreasing those of spermidine and spermine (9, 10).Spermine and spermidine are potent blockers of outward potassium (K ϩ ) currents from inward rectifier K ϩ (Kir) channels (11-13). These channels conduct larger inward currents at membrane voltages below the resting potential than outward currents at more positive voltages. The long, positively charged polyamines, spermine (ϩ4) or spermidine (ϩ3), mediate rectification by binding to negatively charged residues in the channel pore (12,14). The catabolized polyamine putrescine (ϩ2) is less effective at blocking outward flow of K ϩ ions (11, 12). Although Kir channels have been implicated in the regulation of many different physiological processes, including heart rate and membrane excitability (15), there are currently no reports suggesting their involvement in the regulation of cell migration. However, numerous studies have suggested that K ϩ efflux is a critical factor in modulating cell migration (16)(17)(18)(19). In this study, we show that both the catalytic activity of SSAT and the downstream catabolism of polyamines are specifically required for ␣9...

No abstract

The hemidesmosome is a multimolecular complex that integrates the extracellular matrix with the keratin cytoskeleton and that stabilizes epithelial attachment to connective tissue. A 180 kDa protein (BP180, type XVII collagen), first identified by its reactivity with autoantibodies in the serum of patients with a blistering skin disease called bullous pemphigoid (BP), is a transmembrane component of the hemidesmosome with a collagen-like extracellular domain. Here, using recombinantly expressed molecules and the yeast two-hybrid assay, we have identified alpha6 integrin as a BP180-binding partner. The association between specific domains of the BP180 and alpha6 integrin molecules is inhibited by a 14 mer peptide, whose sequence is identical to amino acid residues 506-519 in the noncollagenous region of the ectodomain of the BP180 molecule, as well as by antibodies raised against this peptide. The 14 mer peptide sequence is part of an epitope recognized by autoantibodies that are pathogenic in BP. In vivo, when 804G cells are plated into medium containing the same peptide, they fail to assemble hemidesmosomes. Furthermore, although BP180 and certain cytoplasmic components of the hemidesmosome colocalize in the peptide-treated cells, they are aberrantly distributed and fail to show extensive association with (alpha6beta4 integrin. Taken together, our results indicate that BP180 is a novel transmembrane ligand of the alpha6beta4 integrin heterodimer. In addition, our data provide support for the possibility that BP180 and alpha6 integrin interaction is not only mediated by the BP epitope but is necessary for hemidesmosome formation.

Transdominant inhibition of integrins or integrin-integrin crosstalk is an important regulator of integrin ligand binding and subsequent signaling events that control a variety of cell functions in many tissues. Here we discuss examples of integrin crosstalk and detail our current understanding of the molecular mechanisms that are involved in this receptor phenomenon. The cytoskeleton associated protein talin is a key regulator of integrin crosstalk. We describe how the interaction of talin and the cytoplasmic tail of β integrin is controlled and how competitive inhibitors of this binding play a role in integrin crosstalk. We conclude with a discussion of how integrin crosstalk impacts the interpretation of integrin inhibitor and knockdown studies in both the laboratory and clinical setting.