Determination of N- and C-terminal borders of the transmembrane

domain of integrin subunits.

Running title: Integrin transmembrane segment

Anne Stefansson¶, Annika Armulik¶*, IngMarie Nilsson#, Gunnar von Heijne# and

Staffan Johansson¶

¶Department of Medical Biochemistry and Microbiology

Uppsala University, BMC, Box 582,

SE-751 23 Uppsala, Sweden

#Department of Biochemistry and Biophysics

Stockholm University

SE-106 91 Stockholm, Sweden

* Present address: Department of Cell and Molecular Biology, Medical Nobel

JBC Papers in Press. Published on March 10, 2004 as Manuscript M400771200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Institute, Karolinska Institute SE-171 77, Stockholm, Sweden

E-mail addresses: annika.armulik@cmb.ki.se , gunnar@dbb.su.se ,

ingmarie@dbb.su.se , staffan.johansson@imbim.uu.se, anne.stefansson@imbim.uu.se

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ABSTRACT

Previous studies on the membrane-cytoplasm interphase of human integrin subunits

have shown that a conserved lysine in subunits α2, α5, β1 and β2 is embedded in the

plasma membrane in the absence of interacting proteins (Armulik et al, 1999, J. Biol.

Chem, 274: 37030-4). Using a glycosylation mapping technique, we here show that

α10 and β8, two subunits that deviate significantly from the integrin consensus

sequences in the membrane-proximal region, were found to have the conserved

lysine at a similar position in the lipid bilayer. Thus, this organisation at the C-

terminal end of the transmembrane (TM) domain seems likely to be general for all 24

integrin subunits.

Furthermore, we have determined the N-terminal border of the TM domains of the

α2, α5, α10, β1 and β8 subunits. The TM domain of subunit β8 is found to be 22 amino

acid long, with a second basic residue (Arg 684) positioned just inside the membrane

at the exoplasmic side, whereas the lipid-embedded domains of the other subunits are

longer, varying from 25 (α2) to 29 amino acids (α10). These numbers implicate that

the TM region of the analyzed integrins (except β8) would be tilted or bent in the

membrane.

Integrin signalling by transmembrane conformational change may involve alteration

of the position of the segment adjacent to the conserved lysine. To test the proposed

piston model for signalling, we forced this region at the C-terminal end of the α5 and

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β1 TM domains out of the membrane into the cytosol by replacing Lys-Leu with

Lys-Lys. The mutation was found to not alter the position of the N-terminal end of

the TM domain in the membrane, indicating that the TM domain is not moving as a

piston. Instead the shift results in a shorter and therefore less tilted or bent TM α-

helix.

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ABBREVIATIONS

aa - amino acid

Lep - leader peptidase

MGD - minimal glycosylation distance

OST - oligosaccharyltransferase

TM - transmembrane

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INTRODUCTION

Integrins are heterodimeric receptors composed of an α-subunit non-

covalently associated with a β-subunit. Each subunit has an N-terminal extracellular

domain, a transmembrane (TM) region and a cytoplasmic domain. The human α- and

β-subunits constitute two unrelated protein families of 18 and 8 members,

respectively (1,2).

Integrins mediate cell adhesion to the pericellular matrix and to neighbouring

cells (1). In addition to the anchoring function, ligand binding to integrins generates

intracellular signals required for several cellular processes, including cell migration

and proliferation. The ability to bind ligands is regulated by mechanisms acting on the

cytoplasmic part of the protein, an unusual receptor feature. Integrin activation by

cytoplasmic signals has been shown to involve transmembrane conformational

changes (3,4). Subsequent ligand binding induces further structural rearrangements, as

monitored by exposure of new epitopes, in the extracellular as well as in the

intracellular domains (5,6).

Recently, significant progress has been made in the elucidation of the

mechanisms controlling integrin activation (“inside-out signalling”) and ligand-

induced signalling (“outside-in signalling”). The cytoplasmic protein talin was found

to bind to the membrane-proximal region of the β1-, β2- and β3-subunits and

thereby activates the integrins (7-10). Integrin activation has been shown to require

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separation of the α- and β-subunit cytoplasmic domains from each other (9,11) and

this is presumably the way by which talin activates integrins. In addition, recent

reports have suggested that the TM domains of the subunits mediate integrin

clustering after ligand binding (12). TM domains therefore appear to contribute to

signalling in both directions across the membrane, rather than serving merely to

connect the intra- and extracellular domains. Evidence for important functions of

integrin TM domains is further provided by their high degree of conservation within

the integrin α- and β-protein families, and also between species for individual

subunits.

Several models have been proposed to explain the transmembrane signalling

of integrins. These are based on different types of movements of the TM domains,

such as rotation, tilting, and piston movement (13-17). As a step towards the

identification of the mechanisms used for outside-in and inside-out signalling, we

have in the present study defined the borders of the TM domains from five selected

integrin subunits. This information has allowed us to test the piston model for integrin

α5β1.

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MATERIALS AND METHODS

Enzymes and Chemicals-Unless stated otherwise, enzymes were purchased from

Promega, MBI Fermenta AB and New England Biolab. For PCR the

puReTaqReady-To-GoPCR Beads from Amersham Biosciences were used. PCR primers were

from DNA technology and TAG Copenhagen. DNA manipulations were made using

the TOPO kit from Invitrogen, the Rapid Ligation kit from Roche and the

QuickChangeSite-Directed Mutagenesis Kit from Stratagene. Ribonucleotides, the

cap analogue m7G(5’)ppp(5’)G and [35S]Met were from Amersham Bioscience.

Dithiothreitol, bovine serum albumin, RNasin ribonuclease inhibitor, plasmid

pGEM1, rabbit reticulocyte lysate and amino acid mixture without methionine were

from Promega. Spermidine was from Sigma.

DNA Manipulations-The DNA sequence coding for the region containing the

predicted TM domain of integrin subunits α2, α5, α10, β1 and β8 were amplified by

polymerase chain reaction from corresponding cDNAs. The following primers were

used: for α2, α2TMs (5’-ATGATCACAGAGAAAGCCGAAG-3’) and α2TMas

(5’-ATCATATGTTTTCTTTTGAAG-3’); for α5, α5TMs (5’-

ATGATCACAGAAGGCAGCTATG-3’) and α5TMas (5’-

ATCATATGGGAGCGTTTGAAG-3’); for α10N-terminal, α10N-TMs (5’-

ATGATCACACAGACCCGGCCTATCCT-3’) and α10N-TMas (5’-

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ATCATATGTTTCTTATGGGCAAAGAAGC-3’); for α10C-terminal, α10C-TMs

(5’-TTTATATTGATCACGGTTCAGACCCGGCCTATCC-3’) and α10C-TMas

(5’-ATTTAATCATATGATTTCTTATGGGCAAAGAAGC-3’); for β1, β1TMs

(5’-ATGATCACAGAGTGTCCCACTGG-3’) and β1TMas (5’-

ATCATATGTCTGTCATGAATTATC-3’); for β8N-terminal, β8N-TMs (5’-

TTGATCACTTCAGAATGTTTCTCCAGC-3’) and β8N-TMas (5’-

ATCATATGTATCACCTGTCTAATGATAAGGACTTTAAGC-3’); for β8C-

terminal, β8C-TMs (5’-TTGATCACTTCAGAATGTTTCTCCAGC-3’) and β8C-

TMas (5’-ATCATATGATATCACCTGTCTAATGATAAGGACTTTAAGC-3’).

The sense primers introduced a BclI restriction site and the anti-sense primers an

NdeI restriction site. In the primers β8N-TMas and β8C-TMas a nucleotide was

changed without altering the amino acid sequence to avoid an unwanted BclI

restriction site (marked in bold). The pGEM1-based Lep vectors encoding the protein

leader peptidase (Lep) with a glycosylation acceptor site at different positions have

been described previously (18). The amplified TM regions were cloned into the Lep

vectors, replacing a transmembrane region in the translated leader peptidase (Fig. 1).

For α2 the amino acid residues 1126-1162 were inserted, for α5 residues 992-1029,

for α10 residues 1115-1154, for β1 residues 722-760, and for β8 residues 673-708.

The mutations L1023K in α5 TM and L753K in β1 TM (marked in bold) were

introduced using the following primers: α5L-Ks (5’-

ATCCTCTACAAGAAGGGATTCTTCAAA-3’), α5L-Kas (5’-TTTGAAGAATCCCTTCTTGTAGAG

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β1L-Ks (5’-CTGATTTGGAAAAAGTTAATGATAATT-3’), and β1L-Kas (5’-

AATTATCATTAACTTTTTCCAAATCAG-3’). All constructs were verified by

DNA sequencing (ABI PRISMTM 310 Genetic Analyzer).

Expression in vitro-The Lep vector constructs were transcribed by SP6 RNA

polymerase and translated in reticulocyte lysate in the presence and absence of dog

pancreas microsomes as described (19). Proteins were analyzed by SDS-

polyacrylamide gel electrophoresis, and bands were quantitated on a Fluorescent

Image Reader FLA-3000 phosphoimager using the Image Reader 1.1 software. The

extent of glycosylation of a given construct was calculated as the quotient between the

intensity of the glycosylated band divided by the summed intensities of the

glycosylated and non-glycosylated bands. In general, the glycosylation efficiency

varied by no more than ±5% between different experiments. The “minimal

glycosylation distance” (MGD), i.e. the number of residues between the acceptor site

and the lipid bilayer required to reach half-maximal glycosylation efficiency,

previously determined for poly-Leu TM segments (18) was used to identify the

water-lipid interface.

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RESULTS

Glycosylation mapping

The glycosylation mapping technique has previously been described in detail (18).

Briefly, the assay is based on the ability of the lumenally disposed ER enzyme

oligosaccharyl transferase (OST) (20) to add a glycan to the Asn residue in Asn-X-

Ser/Thr glycosylation acceptor sites in target proteins. The minimal number of

residues between the end of the model transmembrane segment (25LV) composed of

25 consecutive leucines and one valine, and flanked by polar residues and the

acceptor Asn required for half-maximal glycosylation is ~10 residues when the

acceptor site is C-terminal to the TM segment, and ~14 residues when it is N-

terminal to the TM segment (18). Using a glycosylation site scanning approach to

identify the corresponding “minimal glycosylation distance” (MGD) for a TM

segment of interest, one can thus estimate the position of this TM segment in the ER

membrane by comparison with the MGD for the model TM segment (15).

To locate both the N- and C-terminal ends of integrin TM segments relative to the

ER membrane, TM regions of chosen integrin subunits were cloned into two series of

vectors (A and B in Fig. 1) based on the well-characterized integral membrane

protein leader peptidase (Lep). The A-series was used for determining the position of

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the C-terminal end and the B-series for determining the position of the N-terminal

end of the TM segments. The vectors in each series differ only in the position of the

glycosylation acceptor site relative to the TM segment. The constructs were

transcribed and translated in vitro in the presence and absence of dog pancreas rough

microsomes. The measured MGD-values were used to estimate the positions of the

chosen integrin TM segments in the ER membrane by comparison to the 25LV model

TM described above.

Determination of N-terminal borders of the transmembrane domain of integrin

subunits

Five integrin subunits were selected for determination of the N-terminal border of

their TM domains. α2, α5, and β1 were chosen as representatives of α- and β-

subunits with TM domains typical for each of the two protein families, whereas α10

and β8 are examples of interesting deviations from the consensus sequences. For the

analysis, segments of integrin subunits α2 (aa 1126-1162), α5 (aa 992-1029), α10

(aa 1115-1154), β1 (aa 722-760) and β8 (aa 673-708) were inserted into the B-

series vectors (Fig. 1).

The results of in vitro transcriptions/translations of the constructs are summarized in

Fig. 2. As expected, a rapid drop in glycosylation efficiency is seen when the acceptor

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site is moved closer to the TM segment (Fig. 2b, c). For example, the glycosylation

efficiency is reduced from 68% for the α2 TM domain in vector 14 (where the

acceptor Asn is 14 residues upstream of Val1134) to 11% for same TM segment in

vector 13 (where the acceptor Asn is 13 residues upstream of Val1134, Fig. 2c). By

comparison to the MGD-value of 14 residues determined for the model 25LV TM

described above (18), the residue in the α2 TM domain located in the equivalent

position in the membrane as the N-terminal Leu in the model segment is thus

Val1134. We conclude that the N-terminal membrane border is approximately at V1134

for α2, at L999 for α5, at L1123 for α10, at P731 for β1, and at Y682 for β8 (Fig. 4).

Determination of C-terminal borders of the transmembrane domain of integrin

subunits

The C-terminal borders of the TM domains of integrin subunits α2, α5, and β1 have

been determined previously (15). In this study we have determined the C-terminal

border for α10 and β8 subunits. Segments of α10 (aa 1115-1154) and β8 (aa 673-

708) were cloned into the A-series vectors. The glycosylation efficiency of in vitro

expressed proteins was tested as described above. The results are shown in Fig. 3. By

comparison to the MGD value of 10 residues determined for the model 25LV TM

described above (18), the C-terminal membrane border is at A1151 for α10 and at

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I704 for β8 (Fig. 4).

Testing the piston-model.

Several models for integrin-dependent signal transduction across the membrane have

been proposed (13-17), with special attention given to the highly conserved regions

flanking the membrane-cytoplasm interphase. The C-terminal part of the TM domain

has been suggested to move out of the membrane either by sliding of the TM helices

in a piston-like motion (13), by changes in tilt (15), or by a an uncoiling process (14)

(Fig. 6).

We have previously shown that the C-terminal end of the TM segment in β1 shifts

relative to the membrane if a second lysine is introduced next to the conserved

membrane-embedded lysine, i.e., by mutating Leu753 to Lys in β1 (15). In the

present study we analyzed whether the N-terminal end of the TM domain would

move relative to the membrane when the C-terminal end is forced out of the

membrane in this way. The MGD determination was repeated for α5 and β1

constructs carrying the L-to-K mutation at the C-terminal end (α5L1023K and

β1L753K). The results show that no change in the position of the N-terminal end of α5 and

β1 TM domains occurs (Fig. 5). Thus, the luminal N-terminal ends of the α5 and β1

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TM segments do not move relative to the membrane when their C-terminal ends are

forced out of the membrane on the cytoplasmic side.

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DISCUSSION

The role of the cytoplasmic domains of α and β subunits in integrin activation and

signalling is well established (9,21-23). Accumulating data indicate active roles also

for the TM domains (12,24), as well as for the membrane-proximal parts flanking the

TM domains (8,25). However, it is not yet known what kind of molecular movements

of the TM domains are linked to these events. In order to better understand the role of

integrin TM domains we previously determined the membrane-cytoplasm interface

for α2, α5, β1, and β2 using an in vitro glycosylation-mapping assay (15).

Unexpectedly, the transmembrane domain was found to include an additional 5-6

amino acids at the C-terminal end compared to earlier predictions; this result was

subsequently confirmed by NMR studies of the β3 TM and cytoplasmic domains in

dodecylphosphocholine micelles (26). A basic amino acid, which is conserved in all

human integrin subunits residue, Arg in αV and β7 and Lys in all other subunits, is

thus located in the plasma membrane in the absence of interacting proteins. A basic

residue at this position is likely to influence interactions with membrane proteins

and/or the orientation of the TM domain in the lipid bilayer.

In the present study, the characterization of integrin TM domains has been extended

with i) determination of the C-terminal border of β1-associated subunit α10, ii)

determination of the N-terminal borders of α2, α5, α10, β1, iii) determination of both

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ends of the strongly divergent β8 TM domain, and iv) a test of the validity of the

piston model (13) as a possible mechanism for propagating conformational changes

across the plasma membrane.

The amino acid motif (K/R)XGFFKR is present at the membrane-cytoplasm interface

in all 18 integrin α-subunits except α8 (KCGFFDR), α9 (KLGFFRR), α10

(KLGFFAH), and α11 (KLGFFRS). In view of the high degree of conservation of the motif,

minor deviations such as those in α8 and α10 may be functionally significant.

Analysis by the in vitro glycosylation assay showed that the α10 TM domain extends

1-2 amino acid residues further at the C-terminus compared to the TM domain of

other α-subunits. This result is not unexpected considering the absence in α10 of the

strongly charged dipeptide KR. Thus, the membrane-embedded lysine in α10 resides

even deeper inside the membrane than in other α-subunits.

It is not obvious from the primary sequences where the N-terminal borders of integrin

TM domains are located. The border has usually been predicted to be located 23

amino acids or more upstream of the conserved membrane-embedded lysine (e. g.

K752 in β1) (27-31). However, not all integrin subunits may necessarily have TM

domains of identical length, and the α-subunits in particular have variable numbers

of non-polar amino acids upstream of the predicted 23 residues that may influence

the length of the TM segment.

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Applying the in vitro glycosylation method, the N-terminal borders for α5 and α10

were found to be located at the same distance upstream of the membrane-embedded

lysine (Fig. 4). Thus, both these subunits have a tryptophan in position to interact with

the carbonyl group of phospholipids, a commonly found arrangement in membrane

proteins (32). In α5, P998 immediately outside of the TM domain will promote

disruption of the α-helix. Subunit α10 has a weakly polar serine residue at the

corresponding position and a proline located 4 residues further upstream, suggesting

that the α-helix may continue approximately one turn into the extracellular domain.

For the α2 subunit the water-lipid interface was found to reside two residues further

towards the C-terminus than in α5 and α10, if the conserved KLGFF motif is used as

the reference point. Thus in α2, G1133 and P1131 are located approximately one and

three residues outside the membrane, respectively and may serve as helix-breakers.

According to the results of the glycosylation mapping assay, the approximate length

of the membrane spanning segment is 25 residues in α2, 27 residues in α5, and 29

residues in α10. One implication of these results is that the TM α-helix of the three

selected representatives of integrin α-subunits are not running perfectly perpendicular

to the membrane, but rather have to be bent, tilted and/or coiled in slightly different

ways to fit into the membrane.

The TM segment of β1 was found to be approximately 26 aa in length, with P731 at

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the N-terminal border instead of the commonly predicted D728. The TM segment of

β8 was originally suggested to consist either of the 15 hydrophobic amino acids that

are flanked by R684 and K700 at the N- and C-terminus, respectively, or by a 30

amino acid segment (33). Our experimental data indicate that both R684 and K700

are located inside the membrane, resulting in a TM segment of 22 residues in lenght.

Still, the TM segment in β8 is significantly shorter than that in β1. The β8 TM

domain also exhibits several other unique features. The sequence around the

membrane-cytoplasm interface, WKLLXX(I/F)HDRR/KE, is conserved in β1, β3,

β5, and β6, while significant deviations from the motif are present in β2, β7, and β8; β4

shows only weak similarity in this part of the protein, as well as in the cytoplasmic

domain. Our measurements show that the β8 TM domain continues four residues

beyond the membrane-embedded lysine, compared to approximately six residues in

β1. Other notable differences between β8 and β1 are the absence of W in front of the

conserved C-terminal K, and the replacement of HDRRE with another polar

sequence. Furthermore, the membrane-embedded arginine (R684) is only found in

β8, while the β8 TM domain lacks both the glycine and alanine residues that are present

at specific positions in most other β-subunits.

Whether these structural features confer any particular function to β8 is presently not

known. However, β8, as well as β1, β3, β5, and β6, associate with the αV subunit and

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therefore the unusual structure of the β8 TM domain most likely does not influence

the selection of α-subunit partner. Since β8 lacks key talin-binding residues in the

membrane proximal and cytoplasmic domains (10), i.e. IH758, W775 and NPIY783

in human β1, αVβ8 may have a different mechanism of activation than other

integrins. Possibly, this is reflected in the structure of the TM domain. Relatively little

is yet known about the signalling properties of αVβ8, and further studies may clarify

whether the β8 TM domain has any specific role in this context.

Under the conditions of the glycosylation assay, the α-carbon of K752 in the isolated

TM domain of β1 and the corresponding lysine in other integrin α- and β-TM

domains, is clearly located in the lipid bilayer. A similar position for the lysine was

found when a β3-fragment consisting of the TM and cytoplasmic domains was

analysed by NMR spectroscopy (26). However, the presence of a tryptophan or

tyrosine at the position immediately preceding the conserved K/R in all integrin

subunits except β8 suggests that the (W/Y)(K/R) motif may be found at the

membrane-cytoplasm interphase in certain integrin conformation(s). Membrane

proteins commonly have a tryptophan or a tyrosine at the ends of the TM segments

where they can serve as anchors by interacting both with the fatty acid chains and the

carbonyl group of the phospholipids via hydrophobic and hydrogen bonds,

respectively (32). The basic residue (e. g. K752 in β1) may serve as a flexible anchor,

which can interact via its long side chain with the negatively charged phosphate

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groups of phospholipids even if the α-carbon moves a short distance in or out of the

membrane.

It has been suggested that a movement of the conserved C-terminal end of the TM

domain in or out of the membrane could occur if the whole TM helix slides as a rigid

piston through the membrane (13) (Fig. 6a). Since the extracellular region

immediately outside of the TM domains analysed in this study contains a short stretch

of non-polar or weakly polar residues, such a model appeared to be possible.

However, we find that the position of the N-terminal end of the TM helix remains

unaltered when the position of the C-terminal end is forced to shift from FK1027 to

YK1022 for α5, and from IH758 to WK752 for β1, by replacing a leucine with lysine

at positions 1023 and 753, respectively (15). Therefore, the piston model seems

unlikely for the α5 and β1 subunits. If the C-terminal end of the TM domains is

induced to move into the cytoplasm by physiological stimuli, e.g., by a protein-

protein interaction, altered tilting and/or uncoiling seem more likely mechanisms to

account for the shortening of the membrane-spanning segment. Two schematic

models for such shortening of the TM helix is pictured in Fig. 6b, c. Further

experiments will be needed to test whether alterations in the orientation of the

membrane-proximal region of one or both integrin subunits are linked to the active,

inactive, or ligand-stimulated conformations.

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ACKNOWLEDGMENTS

This work was supported by grants from the Swedish Science Council to SJ and GvH,

the Swedish Cancer Foundation to SJ and GvH, and King Gustaf V:s 80-årsfond to

SJ. We thank Drs Evy Lundgren-Åkerlund and Stephen I. Nishimura for providing

cDNA for α10 and β8, Dr Masao Sakaguchi (Fukuoka) for providing dog pancreas

microsomes and Tara Hessa for helpful assistance.

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15, 572-58224. Armulik, A., Velling, T., and Johansson, S. (2004) Mol Biol Cell In press25. Xiong, Y. M., Chen, J., and Zhang, L. (2003) J Immunol 171, 1042-105026. Li, R., Babu, C. R., Valentine, K., Lear, J. D., Wand, A. J., Bennett, J. S., and

DeGrado, W. F. (2002) Biochemistry 41, 15618-1562427. Argraves, W. S., Suzuki, S., Arai, H., Thompson, K., Pierschbacher, M. D.,

and Ruoslahti, E. (1987) J Cell Biol 105, 1183-119028. Camper, L., Hellman, U., and Lundgren-Akerlund, E. (1998) J Biol Chem

273, 20383-2038929. Tuckwell, D. S., and Humphries, M. J. (1997) FEBS Lett 400, 297-30330. Takada, Y., and Hemler, M. E. (1989) J Cell Biol 109, 397-40731. Schneider, D., and Engelman, D. M. (2003) J Biol Chem 32. Killian, J. A., and von Heijne, G. (2000) Trends Biochem Sci 25, 429-43433. Moyle, M., Napier, M. A., and McLean, J. W. (1991) J Biol Chem 266,

19650-19658.

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Figure 1

Orientation of leader peptidase (Lep) and Lep-integrin constructs in microsomes.

Lep constructs used for the determination of the C-terminal (A) and N-terminal end

(B) of integrin TM segments (i-segments). The minimal glycosylation distance

(MGD) is the number of residues between the end of the i-segment and the Asn in the

engineered Asn-Ser-Thr glycosylation acceptor site (*) needed for half-maximal

glycosylation of the protein.

OST, oligosaccharyl transferase

Figure 2

Determination of the N-terminal borders of integrin TM segments.

(A) In vitro translation of B-vector constructs in the presence (+M) and absence (-M)

of dog pancreas microsomes analyzed by SDS-PAGE. (B) Glycosylation efficiencies

based on quantitation of the bands from the polyacrylamide gel as a function of the

number of residues between the N-terminal end of the i-segment and the Asn in the

engineered Asn-Ser-Thr glycosylation acceptor site. (C) Amino acid (aa) sequence

of the alpha2 TM region in three different B-vectors (13-15). Note that the

glycosylation acceptor site (NST, marked in bold) is positioned closer to the TM

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insert in vectors with lower numbers.

Figure 3

Determination of the C-terminal borders of integrin TM segments.

(A) In vitro translation of A-vector constructs in the presence (+M) and absence (-M)

of dog pancreas microsomes analyzed by SDS-PAGE. (B) Glycosylation efficiencies

based on quantitation of the bands from the polyacrylamide gel as a function of the

number of residues between the C-terminal end of the i-segment and the Asn in the

engineered Asn-Ser-Thr glycosylation acceptor site.

Figure 4

Transmembrane segments of integrin α2, α5, α10, β1 and β8.

The determined N-terminal border is approximately at V1134 for α2, at L999 for α5,

at L1123 for α10, at P731 for β1 and at Y682 for β8. The determined C-terminal

border is approximately at A1151 for α10 and at I704 for β8. The C-terminal borders

for α2, α5 and β1 are from Armulik et al. (15) and both borders for the model TM

segment 25LV are from Nilsson et al. (18). Gaps have been inserted in order to align

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the ends of the TM domains. ECS, extracellular space.

Figure 5

Comparison of N-terminal borders of the α5 and β1 TM segment with the borders of

the α5L1023K and β1L753K TM segments.

Glycosylation efficiencies of α5 and α5L1023K was analysed in three different B-

vectors and the same was done for β1 and β1L753K. As seen in the graph, the MGD-

values were not altered by the mutations.

Figure 6

Hypothetical models of transmembrane signalling.

In the piston model (A) the TM regions (red) are assumed to slide as a rigid piston

through the membrane, moving the conserved lysine (K) of one or both subunits in

and out of the membrane. In the coiled model (B) the two TM regions (red) are coiled

around each other as a coiled coil. When uncoiled, the TM α-helices are too long to

run perpendicular to the plasma membrane. Instead the C-terminal end of the TM

region would be moved into the cytoplasm. In the tilting model (C) the TM regions

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adapt to the bilayer by tilting. Changes in the tilt angle will push the C-terminal end

of the TM into the cytosol. The separation of the TM regions after talin binding is not

included in these models since it is not clear in which conformation this occurs. If

model B is correct, the coiled-coil structure would correspond to a conformation

before activation by talin.

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DEKAEVPTGVIIGSIIAGILLL----LALVAILWKLGFFKRKYE 25 aa KAEGSYGVPLWIIILAILFGLLL--LGLLIYILYKLGFFKRSLP 27 aa VQTRPILISLWILIGSVLGGLLLLALLVFCLWKLGFFAHKKIPE 29 aa

a2a5a10

ECPTGPDIIPIVAGVVAGIVLIG---LALLLIWKLLMIIHDRRE 26 aab1TSECFSSPSYLRIFFIIFIVT-------FLIGLLKVLIIRQVIL 22 aab8

Fig.4

CytoplasmECS

TM lenght1164

1031

1157

1125

990

1114

762

709

722

673

LISQQQLLLLLLLLLLLLL---LLLLLLLLLLLLVKKKKH25LV

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12 13 14 15 16 170

20

40

60

80

100Alpha5L1023K

Alpha5

Beta1L753K

�Beta1

Fig.5

Vector

Gly

cosy

latio

n

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K K

Fig.6

PISTON

KK

COILED

K K

TILTING

KKKKKK

A B C

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JohanssonAnne Stefansson, Annika Armulik, IngMarie Nilsson, Gunnar von Heijne and Staffan

subunitsDetermination of N- and C-terminal borders of the transmembrane domain of integrin

published online March 10, 2004J. Biol. Chem. 

  10.1074/jbc.M400771200Access the most updated version of this article at doi:

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