Shape and shift changes related to the function of leukocyte integrins LFA-1 and Mac-1.

Integrin activity on leukocytes is controlled tightly, ensuring that ligand binding occurs only when leukocytes are in contact with their targets. For an integrinlike LFA-1, this ligand-binding activity comes about as a result of increased integrin clustering. Affinity regulation of integrins also plays a role, but the conformational changes giving rise to increased affinity appear to be secondary to clustering. Conformationally altered LFA-1 can be created artificially by deletion of the I domain, which is the key domain involved in ligand binding for many but not all integrins. Although I domain-deleted LFA-1 (DeltaI-LFA-1) cannot bind ligand, it is able to signal constitutively into the cell. One measure of this signaling activity is the ability of DeltaI-LFA-1 to activate beta1 integrins on the same T lymphocyte. Leukocytes use LFA-1 to migrate across the endothelium. Active beta1 integrins may be required subsequently to bind the matrix proteins encountered by leukocytes as they continue their voyage into the tissue interior.

The regulation of integrin function by Ca(2+).

Integrins are metalloproteins whose receptor function is dependent on the interplay between Mg(2+) and Ca(2+). Although the specificity of the putative divalent cation binding sites has been poorly understood, some issues are becoming clearer and this review will focus on the more recent information. The MIDAS motif is a unique Mg(2+)/Mn(2+) binding site located in the integrin alpha subunit I domain. Divalent cation bound at this site has a structural role in coordinating the binding of ligand to the I domain containing integrins. The I-like domain of the integrin beta subunit also has a MIDAS-like motif but much less is known about its cation binding preferences. The N-terminal region of the integrin alpha subunit has been modelled as a beta-propeller, containing three or four 'EF hand' type divalent cation binding motifs for which the function is ill defined. It seems certain that most integrins have a high affinity Ca(2+) site which is critical for alphabeta heterodimer formation, but the location of this site is unknown. Finally intracellular Ca(2+) fluxes activate the Ca(2+) requiring enzyme, calpain, which regulates cluster formation of leucocyte integrins.

From crystal clear ligand binding to designer I domains.

A long awaited crystal structure of an integrin I domain in complex with a peptide derived from collagen has revealed the ligand-bound conformation of this domain and suggests a mechanism for allosteric control of integrin function by ligand binding. Also, a computational protein design approach has allowed the creation of stable, high affinity forms of the I domain for the first time.

Effects of I domain deletion on the function of the beta2 integrin lymphocyte function-associated antigen-1.

A subset of integrin alpha subunits contain an I domain, which is important for ligand binding. We have deleted the I domain from the beta2 integrin lymphocyte function-asssociated antigen-1 (LFA-1) and expressed the resulting non-I domain-containing integrin (DeltaI-LFA-1) in an LFA-1-deficient T cell line. DeltaI-LFA-1 showed no recognition of LFA-1 ligands, confirming the essential role of the I domain in ligand binding. Except for I domain monoclonal antibodies (mAbs), DeltaI-LFA-1 was recognized by a panel of anti-LFA-1 mAbs similarly to wild-type LFA-1. However, DeltaI-LFA-1 had enhanced expression of seven mAb epitopes that are associated with beta2 integrin activation, suggesting that it exhibited an "active" conformation. In keeping with this characteristic, DeltaI-LFA-1 induced constitutive activation of alpha4beta1 and alpha5beta1, suggesting intracellular signaling to these integrins. This "cross-talk" was not due to an effect on beta1 integrin affinity. However, the enhanced activity was susceptible to inhibition by cytochalasin D, indicating a role for the cytoskeleton, and also correlated with clustering of beta1 integrins. Thus, removal of the I domain from LFA-1 created an integrin with the hallmarks of a constitutively active receptor mediating signals into the cell. These findings suggest a key role for the I domain in controlling integrin activity.

The I domain of integrin leukocyte function-associated antigen-1 is involved in a conformational change leading to high affinity binding to ligand intercellular adhesion molecule 1 (ICAM-1).

On T cells the leukocyte integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg2+ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 alpha subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg2+-induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg2+-activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg2+-activated LFA-1 to ICAM-1 is blocked by peptides covering the alpha4-beta3 loop, the beta3-alpha5 loop, and the alpha5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the beta-propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.

Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.

Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.

Swimming navigation, open-field activity, and extrapolation behavior of two inbred mouse strains with Robertsonian translocation of chromosomes 8 and 17.

Female mice from inbred strains carrying a Robertsonian translocation (nine CBARb and eight C57BL/6Rb) were compared with animals from their respective strains (seven CBA and nine C57BL/6) first in open-field activity (two exposures of 10-min duration), then during 5 days (with six trials each) in Morris' swimming navigation test, and finally, in their ability to extrapolate the future position of a food reward being moved slowly out of their reach. ANOVA (strain and translocation) revealed significant effects of Robertsonian translocations (Rb) in swimming navigation, Rb mice being impaired primarily in the initial phases of acquisition and during the first trials of platform reversal and the impairment being stronger in C57BL/6 mice. In the open field, Rb mice were as active as the normal strains but showed significantly increased path tortuosity and moved slightly faster. In the extrapolation task, Rb mice showed above-chance levels in moving to the target indicated by the disappearance of the stimulus, while normal mice chose at chance levels, but the translocation effects were not statistically significant. These data indicate that telocentric fusion of chromosomes may entail behavioral alterations, perhaps by subtle changes in neurotransmitters or limbic circuitry. The expression of such alterations, however, can be remarkably strain dependent.

Tagging secretory and membrane proteins with a tyrosine sulfation site. Tyrosine sulfation precedes galactosylation and sialylation in COS-7 cells.

Sulfation of proteins on tyrosines is a late Golgi modification that can be used to label proteins with [35S]sulfate for the analysis of post-Golgi transport. To extend the use of this modification to proteins not naturally sulfated, we fused a tyrosine sulfation site, the carboxyl-terminal nonapeptide of cholecystokinin precursor, to the carboxyl terminus of two normally unsulfated proteins: alpha 1-proteinase inhibitor, a secretory protein, and subunit H1 of the asialoglycoprotein receptor; a type II membrane protein. The tagged proteins were efficiently sulfated in transfected COS-7 and Madin-Darby canine kidney cells. Specifically in COS-7 cells, the proteins were sulfated before they were galactosylated and sialylated and were converted to the mature forms with a half-time of approximately 2-3 min. This is in contrast to other cell types in which tyrosine sulfation was found to be virtually the last modification of the Golgi apparatus. Our results suggest that tyrosine sulfation occurs before the trans-Golgi in transfected COS-7 cells.

Protein EnvM is the NADH-dependent enoyl-ACP reductase (FabI) of Escherichia coli.

The EnvM protein was purified from an overproducing Escherichia coli strain. It showed NADH-dependent enoyl-acyl carrier protein (ACP) reductase activity using both crotonyl-ACP and crotonyl-CoA as substrates. The protein bound a radioactive diazaborine derivative in the presence of NAD+ and radioactive NAD+ in the presence of the drug. Based on these data, it is concluded that EnvM is the NADH-dependent enoyl-ACP reductase (EC 1.3.1.9) of E. coli and we propose to rename the corresponding gene fabI.

Related signals for endocytosis and basolateral sorting of the asialoglycoprotein receptor.

The major subunit of the human asialoglycoprotein receptor contains signals for efficient endocytosis and specific basolateral expression in polarized Madin-Darby canine kidney cells, both of which are located within its 40-residue cytoplasmic domain. The aromatic residue in this segment, tyrosine 5, which is necessary for efficient clustering into clathrin-coated pits at the plasma membrane, is also necessary for exclusive basolateral delivery. Mutation of this residue to alanine resulted in a nonpolar expression of the protein. Replacement of tyrosine 5 with phenylalanine yielded almost wild-type rates of endocytosis as well as specific basolateral expression, indicating that tyrosine phosphorylation is not essential for either sorting step. The close similarity between the two sorting signals was further corroborated by deletion mutants showing that the amino-terminal 10 residues of the cytoplasmic domain are sufficient for basolateral polarity and efficient endocytosis. The kinetics of appearance of newly synthesized wild-type and mutant receptor protein at the apical and basolateral surfaces indicate that these proteins are sorted intracellularly and are transported directly to the respective domains. Mutants affected in basolateral sorting lost polarity, i.e, appeared to similar extents on both surfaces, indicating that there is no significant apical sorting information elsewhere in the protein. The close correlation between endocytosis and basolateral polarity suggests common recognition mechanisms at the plasma membrane and in the trans-Golgi network.