Limits...
The lymphocyte function-associated antigen 1 I domain is a transient binding module for intercellular adhesion molecule (ICAM)-1 and ICAM-3 in hydrodynamic flow.

Knorr R, Dustin ML - J. Exp. Med. (1997)

Bottom Line: The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes.Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity.This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.

View Article: PubMed Central - PubMed

Affiliation: Center for Immmunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
The I domain of lymphocyte function-associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.

Show MeSH

Related in: MedlinePlus

The distance of the I domain from the bilayer membrane does not result in a stable interaction.  (A) Schematic of I-EF and I-CD2. In I-EF, the I domain was extended with repeats III, IV, V, VI, and the  first few amino acids of repeat VII of the LFA-1 α  chain. I-EF is linked to the Decay Accelerating Factor– GPI-anchoring signal as in I-GPI. In I-CD2, the spacer  between the I domain and the Decay Accelerating Factor–GPI-anchoring signal consists of a few amino acids  of the domain 1, the linker region, domain 2, and the  stalk of human CD2. (B) Interaction of COS-1 cells  transiently transfected with I-GPI, I-CD2, or I-EF, on  membranes containing 500 molecules/μm2 ICAM-1 in  HBS/BSA containing 2 mM MgCl2. The data were  normalized according to a FACS® analysis and are presented as percent transfectants. The rolling interaction  was statistically significant with P <0.01 for each construct. (C) The rolling velocity of COS-1 cells transiently transfected with I-GPI, I-CD2, or I-EF on  membranes containing 500 molecules/μm2 ICAM-1 in  HBS/BSA buffer containing 2 mM MgCl2 was determined.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199009&req=5

Figure 7: The distance of the I domain from the bilayer membrane does not result in a stable interaction. (A) Schematic of I-EF and I-CD2. In I-EF, the I domain was extended with repeats III, IV, V, VI, and the first few amino acids of repeat VII of the LFA-1 α chain. I-EF is linked to the Decay Accelerating Factor– GPI-anchoring signal as in I-GPI. In I-CD2, the spacer between the I domain and the Decay Accelerating Factor–GPI-anchoring signal consists of a few amino acids of the domain 1, the linker region, domain 2, and the stalk of human CD2. (B) Interaction of COS-1 cells transiently transfected with I-GPI, I-CD2, or I-EF, on membranes containing 500 molecules/μm2 ICAM-1 in HBS/BSA containing 2 mM MgCl2. The data were normalized according to a FACS® analysis and are presented as percent transfectants. The rolling interaction was statistically significant with P <0.01 for each construct. (C) The rolling velocity of COS-1 cells transiently transfected with I-GPI, I-CD2, or I-EF on membranes containing 500 molecules/μm2 ICAM-1 in HBS/BSA buffer containing 2 mM MgCl2 was determined.

Mentions: Assuming that the I domain is situated in the globular head of the integrin detected in electron microscopic studies (38), the I domain in intact LFA-1 may protrude ∼14 nm from the the plasma membrane. In I-GPI, this distance is only ∼7 nm. The proximity to the plasma membrane might have a negative effect on the formation of stable bonds. To assess the effect of close spacing between the I domain and the plasma membrane on the rolling interaction, we designed two hybrid molecules, in which a spacer domain is inserted between the I domain and the GPI anchor. In one hybrid protein, the spacer consists of the stalk, the immunoglobulin superfamily (IgSF) domain 2, the linker, and 6 aa of the adjacent IgSF domain 1 of human CD2 (Fig. 7 A). According to the crystal structure of CD2 (39), the I domain should face upwards in this chimeric protein. In the other hybrid protein, the spacer comprised the repeats III, IV, V, VI, and 10aa of repeat VII of CD11a (Fig. 7 A). Repeats I–VII of the integrin α subunit were predicted to fold into a β-propeller domain with the I domain sitting on the upper face of it (40). The I-EF construct would only contain part of the β-propeller domain. The folding and the extensions of this construct and the relative position of the I domain therefore remain unclear. These chimeric proteins were termed I-CD2 and I-EF, respectively. I-CD2 and I-EF were transiently expressed on the surface of COS-1 cells (data not shown). As a control, I-GPI DNA was subcloned in the same expression vector and also transiently expressed in COS-1 cells. To determine the transfection levels of the different constructs, a FACS® analysis was performed in parallel to each flow cell experiment. The data from the flow cell experiments were normalized in respect to the expression levels of the various transfectants.


The lymphocyte function-associated antigen 1 I domain is a transient binding module for intercellular adhesion molecule (ICAM)-1 and ICAM-3 in hydrodynamic flow.

Knorr R, Dustin ML - J. Exp. Med. (1997)

The distance of the I domain from the bilayer membrane does not result in a stable interaction.  (A) Schematic of I-EF and I-CD2. In I-EF, the I domain was extended with repeats III, IV, V, VI, and the  first few amino acids of repeat VII of the LFA-1 α  chain. I-EF is linked to the Decay Accelerating Factor– GPI-anchoring signal as in I-GPI. In I-CD2, the spacer  between the I domain and the Decay Accelerating Factor–GPI-anchoring signal consists of a few amino acids  of the domain 1, the linker region, domain 2, and the  stalk of human CD2. (B) Interaction of COS-1 cells  transiently transfected with I-GPI, I-CD2, or I-EF, on  membranes containing 500 molecules/μm2 ICAM-1 in  HBS/BSA containing 2 mM MgCl2. The data were  normalized according to a FACS® analysis and are presented as percent transfectants. The rolling interaction  was statistically significant with P <0.01 for each construct. (C) The rolling velocity of COS-1 cells transiently transfected with I-GPI, I-CD2, or I-EF on  membranes containing 500 molecules/μm2 ICAM-1 in  HBS/BSA buffer containing 2 mM MgCl2 was determined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199009&req=5

Figure 7: The distance of the I domain from the bilayer membrane does not result in a stable interaction. (A) Schematic of I-EF and I-CD2. In I-EF, the I domain was extended with repeats III, IV, V, VI, and the first few amino acids of repeat VII of the LFA-1 α chain. I-EF is linked to the Decay Accelerating Factor– GPI-anchoring signal as in I-GPI. In I-CD2, the spacer between the I domain and the Decay Accelerating Factor–GPI-anchoring signal consists of a few amino acids of the domain 1, the linker region, domain 2, and the stalk of human CD2. (B) Interaction of COS-1 cells transiently transfected with I-GPI, I-CD2, or I-EF, on membranes containing 500 molecules/μm2 ICAM-1 in HBS/BSA containing 2 mM MgCl2. The data were normalized according to a FACS® analysis and are presented as percent transfectants. The rolling interaction was statistically significant with P <0.01 for each construct. (C) The rolling velocity of COS-1 cells transiently transfected with I-GPI, I-CD2, or I-EF on membranes containing 500 molecules/μm2 ICAM-1 in HBS/BSA buffer containing 2 mM MgCl2 was determined.
Mentions: Assuming that the I domain is situated in the globular head of the integrin detected in electron microscopic studies (38), the I domain in intact LFA-1 may protrude ∼14 nm from the the plasma membrane. In I-GPI, this distance is only ∼7 nm. The proximity to the plasma membrane might have a negative effect on the formation of stable bonds. To assess the effect of close spacing between the I domain and the plasma membrane on the rolling interaction, we designed two hybrid molecules, in which a spacer domain is inserted between the I domain and the GPI anchor. In one hybrid protein, the spacer consists of the stalk, the immunoglobulin superfamily (IgSF) domain 2, the linker, and 6 aa of the adjacent IgSF domain 1 of human CD2 (Fig. 7 A). According to the crystal structure of CD2 (39), the I domain should face upwards in this chimeric protein. In the other hybrid protein, the spacer comprised the repeats III, IV, V, VI, and 10aa of repeat VII of CD11a (Fig. 7 A). Repeats I–VII of the integrin α subunit were predicted to fold into a β-propeller domain with the I domain sitting on the upper face of it (40). The I-EF construct would only contain part of the β-propeller domain. The folding and the extensions of this construct and the relative position of the I domain therefore remain unclear. These chimeric proteins were termed I-CD2 and I-EF, respectively. I-CD2 and I-EF were transiently expressed on the surface of COS-1 cells (data not shown). As a control, I-GPI DNA was subcloned in the same expression vector and also transiently expressed in COS-1 cells. To determine the transfection levels of the different constructs, a FACS® analysis was performed in parallel to each flow cell experiment. The data from the flow cell experiments were normalized in respect to the expression levels of the various transfectants.

Bottom Line: The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes.Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity.This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.

View Article: PubMed Central - PubMed

Affiliation: Center for Immmunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
The I domain of lymphocyte function-associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.

Show MeSH
Related in: MedlinePlus