How Do You Know if N Terminal Is Extracellular

Previous studies on the membrane-cytoplasm interphase of homo 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, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030–37034). Using a glycosylation mapping technique, we here show that α_ten and β₈, ii subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were institute to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-last end of the transmembrane (TM) domain seems probable to be full general for all 24 integrin subunits. Furthermore, we accept adamant the Northward-final border of the TM domains of the α₂, α_v, α_ten, β₁, and β₈ subunits. The TM domain of subunit β₈ is found to be 22 amino acids long, with a second basic residuum (Arg⁶⁸⁴) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (α₂) to 29 amino acids (α_ten). These numbers implicate that the TM region of the analyzed integrins (except β₈) would be tilted or aptitude in the membrane. Integrin signaling 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 signaling, we forced this region at the C-terminal end of the α₅ and 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 finish of the TM domain in the membrane, indicating that the TM domain is not moving every bit a piston. Instead the shift results in a shorter and therefore less tilted or bent TM α-helix.

Integrins are heterodimeric receptors composed of an α subunit noncovalently associated with a β subunit. Each subunit has an N-last extracellular domain, a transmembrane (TM)

 i

The abbreviations used are: TM, transmembrane; aa, amino acids; MGD, minimal glycosylation distance.

1 The abbreviations used are: TM, transmembrane; aa, amino acids; MGD, minimal glycosylation distance.

region and a cytoplasmic domain. The human α- and β subunits constitute two unrelated protein families of 18 and 8 members, respectively (

,

).

Integrins mediate cell adhesion to the pericellular matrix and to neighboring cells (

). In add-on to the anchoring function, ligand bounden to integrins generates intracellular signals required for several cellular processes, including cell migration and proliferation. The ability to bind ligands is regulated past mechanisms interim on the cytoplasmic office of the protein, an unusual receptor feature. Integrin activation past cytoplasmic signals has been shown to involve transmembrane conformational changes (

,

). Subsequent ligand binding induces further structural rearrangements, as monitored past exposure of new epitopes, in the extracellular as well as in the intracellular domains (

,

).

Recently, significant progress has been made in the elucidation of the mechanisms controlling integrin activation ("within-out signaling") and ligand-induced signaling ("outside-in signaling"). The cytoplasmic poly peptide talin was found to bind to the membrane-proximal region of the β₁, β₂, and β_iii subunits and thereby activates the integrins (

,

,

,

). Integrin activation has been shown to require separation of the α and β subunit cytoplasmic domains from each other (

,

), and this is presumably the way by which talin activates integrins. In addition, recent reports take suggested that the TM domains of the subunits mediate integrin clustering later on ligand binding (

). TM domains therefore appear to contribute to signaling in both directions beyond the membrane rather than serving merely to connect the intra- and extracellular domains. Testify for the important functions of integrin TM domains is further provided by their high degree of conservation within the integrin α- and β-protein families and as well between species for private subunits.

Several models have been proposed to explain the transmembrane signaling of integrins. These are based on different types of movements of the TM domains, such as rotation, tilting, and piston movement (

,

,

,

,

). Equally a footstep toward the identification of the mechanisms used for exterior-in and inside-out signaling, nosotros have in the present written report defined the borders of the TM domains from five selected integrin subunits. This information has allowed us to examination the piston model for integrin α_vβ₁.

MATERIALS AND METHODS

Enzymes and Chemicals—Unless stated otherwise, the enzymes were purchased from Promega, MBI Fermenta AB and New England Biolab. For PCR puReTaq™Ready-To-Become™PCR chaplet from Amersham Biosciences were used. PCR primers were from DNA Technology and TAG Copenhagen. Deoxyribonucleic acid manipulations were made using the TOPO kit from Invitrogen, the Rapid Ligation kit from Roche Applied Sciences, and the QuikChange™ site-directed mutagenesis kit from Stratagene. Ribonucleotides, the cap analogue m7G(5′)ppp(5′)One thousand, and [³⁵S] Met were from Amersham Biosciences. 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.

Deoxyribonucleic acid Manipulations—The Deoxyribonucleic acid sequence coding for the region containing the predicted TM domain of integrin subunits α₂, α₅, α₁₀, β_ane, and β₈ were amplified by polymerase chain reaction from corresponding cDNAs. The post-obit primers were used: for α₂, α2TMs (5′-ATGATCACAGAGAAAGCCGAAG-three′) and α2TMas (5′-ATCATATGTTTTCTTTTGAAG-3′); for α₅, α5TMs (5′-ATGATCACAGAAGGCAGCTATG-three′) and α5TMas (5′-ATCATATGGGAGCGTTTGAAG-three′); for α_ten North-concluding, α10N-TMs (5′-ATGATCACACAGACCCGGCCTATCCT-3′) and α10N-TMas (5′-ATCATATGTTTCTTATGGGCAAAGAAGC-3′); for α₁₀ C-terminal, α10C-TMs (5′-TTTATATTGATCACGGTTCAGACCCGGCCTATCC-3′) and α10C-TMas (v′-ATTTAATCATATGATTTCTTATGGGCAAAGAAGC-3′); for β_ane, β1TMs (5′-ATGATCACAGAGTGTCCCACTGG-3′) and β1TMas (five′-ATCATATGTCTGTCATGAATTATC-3′); for β_eight N-terminal, β8N-TMs (5′-TTGATCACTTCAGAATGTTTCTCCAGC-3′) and β8N-TMas (5′-ATCATATGTATCACCTGTCTAATGATAAGGACTTTAAGC-three′); and for β_viii C-last, β8C-TMs (5′-TTGATCACTTCAGAATGTTTCTCCAGC-3′) and β8C-TMas (v′-ATCATATGATATCACCTGTCTAATGATAAGGACTTTAAGC-3′). The sense primers introduced a BclI restriction site and the antisense 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 type). The pGEM1-based Lep vectors encoding the protein Lep (leader peptidase) with a glycosylation acceptor site at different positions have been described previously (

). The amplified TM regions were cloned into the Lep vectors, replacing a transmembrane region in the translated leader peptidase (see Fig. 1). For α₂ the amino acid residues 1126–1162 were inserted, for α₅ residues 992–1029 were inserted, for α₁₀ residues 1115–1154 were inserted, for β₁ residues 722–760 were inserted, and for β₈ residues 673–708 were inserted.

Fig. one Orientation of Lep and Lep-integrin constructs in microsomes. Lep constructs used for the conclusion of the C-terminal (A) and North-terminal ends (B) of integrin TM segments (boxes labeled i). The MGD is the number of residues between the end of the integrin TM segment and the Asn in the engineered Asn-Ser-Thr glycosylation acceptor site (*) needed for half-maximal glycosylation of the protein. OST, oligosaccharyl transferase

• View Large Image
• Effigy Viewer
• Download (PPT)

The mutations L1023K in α_five TM and L753K in β₁ TM (marked in assuming type) were introduced using the following primers: α5L-Ks (5′-ATCCTCTACAAGAAGGGATTCTTCAAA-3′), α5L-Kas (v′-TTTGAAGAATCCCTTCTTGTAGAGGAT-iii′), β1L-Ks (5′-CTGATTTGGAAAAAGTTAATGATAATT-3′), and β1L-Kas (v′-AATTATCATTAACTTTTTCCAAATCAG-3′). All of the constructs were verified by DNA sequencing (ABI PRISM™ 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 domestic dog pancreas microsomes as described (

). The proteins were analyzed past SDS-polyacrylamide gel electrophoresis, and the bands were quantitated by phosphorimaging on a Fluorescent Prototype Reader FLA-3000 using the Image Reader i.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 nonglycosylated bands. In full general, the glycosylation efficiency varied by no more than than ±5% betwixt unlike experiments. The "minimal glycosylation distance" (MGD), i.east. the number of residues betwixt the acceptor site and the lipid bilayer required to achieve one-half-maximal glycosylation efficiency, previously determined for poly-Leu TM segments (

) was used to place the water-lipid interface.

RESULTS

Glycosylation Mapping—The glycosylation mapping technique has previously been described in detail (

). Briefly, the analysis is based on the power of the lumenally disposed endoplasmic reticulum enzyme oligosaccharyl transferase (

) to add a glycan to the Asn residue in Asn-Xaa-(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 sequent 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-concluding to the TM segment and ∼fourteen residues when information technology is Due north-last to the TM segment (

). Using a glycosylation site scanning arroyo to place the corresponding MGD for a TM segment of interest, one tin can thus guess the position of this TM segment in the endoplasmic reticulum membrane by comparing with the MGD for the model TM segment (

).

To locate both the Due north- and C-terminal ends of integrin TM segments relative to the endoplasmic reticulum membrane, TM regions of called integrin subunits were cloned into ii serial of vectors (boxes A and B in Fig. 1) based on the well characterized integral membrane protein Lep. The A series was used for determining the position of the C-concluding end, and the B series was used for determining the position of the Due north-terminal stop of the TM segments. The vectors in each serial 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 endoplasmic reticulum membrane past comparison with the 25LV model TM described above.

Decision of N-concluding Borders of the Transmembrane Domain of Integrin Subunits—Five integrin subunits were selected for determination of the North-last edge of their TM domains. α₂, α₅, and β₁ were chosen every bit representatives of α and β subunits with TM domains typical for each of the two protein families, whereas α^x and β₈ are examples of interesting deviations from the consensus sequences. For the analysis, segments of integrin subunits α_two (aa 1126–1162), α₅ (aa 992–1029), α₁₀ (aa 1115–1154), β₁ (aa 722–760), and β_eight (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 site is moved closer to the TM segment (Fig. 2, B and C). For example, the glycosylation efficiency is reduced from 68% for the α_ii TM domain in vector 14 (where the acceptor Asn is fourteen residues upstream of Val¹¹³⁴) to xi% for same TM segment in vector 13 (where the acceptor Asn is xiii residues upstream ofVal¹¹³⁴; Fig. 2C). By comparison with the MGD value of 14 residues adamant for the model 25LV TM described above (

), the residue in the α_ii 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 Val¹¹³⁴ for α_ii, at Leu⁹⁹⁹ for α_five, at Leu¹¹²³ for α₁₀, at Pro⁷³¹ for β₁, and at Tyr⁶⁸² for β₈ (Fig. 3).

Fig. two Determination of the Northward-final borders of integrin TM segments. A, in vitro translation of B vector constructs in the presence (+M) and absenteeism (-M) of dog pancreas microsomes analyzed by SDS-PAGE. B, glycosylation efficiencies based on quantitation of the bands from the polyacrylamide gel equally a function of the number of residues betwixt the N-last terminate of the integrin TM segment and the Asn in the engineered Asn-Ser-Thr glycosylation acceptor site. C, the amino acid sequence of the α₂ TM region in three different B vectors (, , ). Note that the glycosylation acceptor site (NST, marked in bold type) is positioned closer to the TM insert in vectors with lower numbers.

• View Large Image
• Figure Viewer
• Download (PPT)

Fig. 3 Transmembrane segments of integrin α₂, α₅, α₁₀, β₁, and β₈. The adamant N-terminal edge is approximately at Val¹¹³⁴ for α_two, at Leu⁹⁹⁹ for α₅, at Leu¹¹²³ for α_ten, at Pro⁷³¹ for β₁, and at Tyr⁶⁸² for β_viii. The adamant C-final border is approximately at Ala¹¹⁵¹ for α₁₀ and at Ile⁷⁰⁴ for β_viii. The C-terminal borders for α₂, α₅, and β₁ are from Armulik et al. (), and both borders for the model TM segment 25LV are from Nilsson et al. (). Gaps have been inserted to align the ends of the TM domains. ECS, extracellular space.

• View Large Prototype
• Effigy Viewer
• Download (PPT)

Decision of C-last Borders of the Transmembrane Domain of Integrin Subunits—The C-last borders of the TM domains of integrin subunits α₂, α₅, and β_ane take been adamant previously (

). In this study we have determined the C-final border for the α₁₀ and β₈ subunits. Segments of α₁₀ (aa 1115–1154) and β_eight (aa 673–708) were cloned into the A series vectors. The glycosylation efficiency of in vitro expressed proteins was tested every bit described above. The results are shown in Fig. 4. By comparison with the MGD value of ten residues determined for the model 25LV TM described above (

), the C-terminal membrane border is at Ala¹¹⁵¹ for α₁₀ and at Ile⁷⁰⁴ for β₈ (Fig. iii).

Fig. 4 Determination of the C-terminal borders of integrin TM segments. A, in vitro translation of A vector constructs in the presence (+M) and absenteeism (-M) of dog pancreas microsomes analyzed past SDS-Folio. B, glycosylation efficiencies based on quantitation of the bands from the polyacrylamide gel as a part of the number of residues between the C-last finish of the integrin TM segment and the Asn in the engineered Asn-Ser-Thr glycosylation acceptor site.

• View Large Image
• Effigy Viewer
• Download (PPT)

Testing the Piston Model—Several models for integrin-dependent signal transduction across the membrane accept been proposed (

,

,

,

,

), with special attention given to the highly conserved regions flanking the membrane-cytoplasm interphase. The C-last part of the TM domain has been suggested to move out of the membrane either past sliding of the TM helices in a piston-like move (

), by changes in tilt (

), or by a an uncoiling procedure (

) (see Fig. 6).

Fig. 6 Hypothetical models of transmembrane signaling. In the piston model (A) the TM regions (red) are causeless to slide equally 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 effectually 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 cease of the TM region would exist moved into the cytoplasm. In the tilting model (C) the TM regions arrange to the bilayer by tilting. Changes in the tilt angle will push the C-terminal finish of the TM into the cytosol. The separation of the TM regions after talin binding is non included in these models because it is not clear in which conformation this occurs. If model B is correct, the coiled coil structure would represent to a conformation before activation by talin.

• View Large Image
• Figure Viewer
• Download (PPT)

Nosotros take previously shown that the C-concluding end of the TM segment in β_i 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 (

). In the nowadays written report we analyzed whether the Northward-terminal end of the TM domain would movement relative to the membrane when the C-terminal end is forced out of the membrane in this way. The MGD decision was repeated for α_five and β₁ constructs carrying the L-to-K mutation at the C-terminal cease (α₅L1023K and β_iL753K). The results show that no change in the position of the N-last end of α₅ and β₁ TM domains occurs (Fig. 5). Thus, the luminal N-last ends of the α_v and β₁ TM segments exercise not motion relative to the membrane when their C-terminal ends are forced out of the membrane on the cytoplasmic side.

Fig. 5 Comparing of N-final borders of the α₅ and β₁ TM segment with the borders of the α5L1023K and β1L753K TM segments. Glycosylation efficiencies of α_five and α5L1023K was analyzed in three different B vectors, and the aforementioned was done for β₁ and β1L753K. Every bit seen in the graph, the MGD values were not altered by the mutations.

• View Large Paradigm
• Figure Viewer
• Download (PPT)

DISCUSSION

The role of the cytoplasmic domains of α and β subunits in integrin activation and signaling is well established (

,

,

,

). Accumulating data indicate agile roles also for the TM domains (

,

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

,

). Yet, it is not yet known what kind of molecular movements of the TM domains are linked to these events. To meliorate understand the role of integrin TM domains, nosotros previously determined the membrane-cytoplasm interface for α₂, α₅, β₁, and β_ii using an in vitro glycosylation mapping assay (

). Unexpectedly, the transmembrane domain was constitute to include an additional 5–six amino acids at the C-terminal finish compared with before predictions; this result was later confirmed past NMR studies of the β_iii TM and cytoplasmic domains in dodecylphosphocholine micelles (

). A basic amino acid, which is conserved in all human integrin subunits residue, Arg in α_V and β₇ and Lys in all other subunits, is thus located in the plasma membrane in the absenteeism of interacting proteins. A bones 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) conclusion of the C-terminal edge of β1-associated subunit α_x; (ii) determination of the N-terminal borders of α₂, α₅, α₁₀, and β_ane; (iii) determination of both ends of the strongly divergent β₈ TM domain; and (iv) a test of the validity of the piston model (

) as a possible mechanism for propagating conformational changes across the plasma membrane.

The amino acid motif (One thousand/R)XGFFKR is nowadays at the membrane-cytoplasm interface in all xviii integrin α subunits except α₈ (KCGFFDR), α_nine (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 α_x may be functionally significant. Analysis past the in vitro glycosylation assay showed that the α₁₀ TM domain extends ane–two amino acid residues further at the C terminus compared with the TM domain of other α subunits. This result is not unexpected because the absence in α₁₀ of the strongly charged dipeptide KR. Thus, the membrane-embedded lysine in α₁₀ resides fifty-fifty deeper within 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 (eastward.1000. Lys⁷⁵² in β_i) (

,

,

,

,

). Yet, non all integrin subunits may necessarily take TM domains of identical length, and the α subunits in particular have variable numbers of nonpolar amino acids upstream of the predicted 23 residues that may influence the length of the TM segment.

Applying the in vitro glycosylation method, the Due north-last borders for α_v and α₁₀ were found to be located at the same distance upstream of the membrane-embedded lysine (Fig. 3). Thus, both these subunits have a tryptophan in position to collaborate with the carbonyl group of phospholipids, a commonly plant arrangement in membrane proteins (

). In α_v, Pro⁹⁹⁸ immediately outside of the TM domain will promote disruption of the α-helix. Subunit α_x has a weakly polar serine residue at the corresponding position and a proline located four residues further upstream, suggesting that the α-helix may continue approximately 1 turn into the extracellular domain. For the α₂ subunit the water-lipid interface was found to reside 2 residues farther toward the C terminus than in α₅ and α₁₀, if the conserved KLGFF motif is used equally the reference point. Thus, in α₂, Gly¹¹³³ and Pro¹¹³¹ are located approximately 1 and 3 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 α_two, 27 residues in α_five, 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 non running perfectly perpendicular to the membrane but rather have to exist aptitude, tilted, and/or coiled in slightly different ways to fit into the membrane.

The TM segment of β₁ was found to be ∼26 aa in length, with Pro⁷³¹ at the N-terminal border instead of the unremarkably predicted Asp⁷²⁸. The TM segment of β₈ was originally suggested to consist either of the fifteen hydrophobic amino acids that are flanked past Arg⁶⁸⁴ and Lys⁷⁰⁰ at the Due north and C termini, respectively, or past a xxx-amino acid segment (

). Our experimental information betoken that both Arg⁶⁸⁴ and Lys⁷⁰⁰ are located within the membrane, resulting in a TM segment of 22 residues in length. Yet, the TM segment in β_viii 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)HDR(R/Grand)E, is conserved in β₁, β₃, β_five, and β_{half dozen}, whereas significant deviations from the motif are present in β₂, β₇, and β₈; β₄ shows just weak similarity in this office of the protein, as well as in the cytoplasmic domain. Our measurements bear witness that the β_viii TM domain continues four residues beyond the membrane-embedded lysine, compared with approximately half dozen residues in β₁. Other notable differences between β_eight and β₁ are the absence of Trp in front of the conserved C-terminal Lys, and the replacement of HDRRE with another polar sequence. Furthermore, the membrane-embedded arginine (Arg⁶⁸⁴) is only found in β₈, whereas the β_viii 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 part to β₈ is soon not known. However, β₈, as well as β_one, β_three, β₅, and β₆, associate with the α_V subunit, and therefore the unusual construction of the β₈ TM domain most likely does not influence the selection of the α subunit partner. Because β₈ lacks fundamental talin-bounden residues in the membrane proximal and cytoplasmic domains (

), i.e. Ile-His⁷⁵⁸, Trp⁷⁷⁵, and Asn-Pro-Ile-Tyr⁷⁸³ in human β₁, α_Vβ₈ 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 signaling properties of α_Vβ_eight, and farther studies may clarify whether the β_viii TM domain has any specific role in this context.

Nether the conditions of the glycosylation assay, the α-carbon of Lys⁷⁵² 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 like position for the lysine was plant when a β₃ fragment consisting of the TM and cytoplasmic domains was analyzed by NMR spectroscopy (

). However, the presence of a tryptophan or tyrosine at the position immediately preceding the conserved Lys/Arg in all integrin subunits except β₈ suggests that the (West/Y)(K/R) motif may be plant at the membrane-cytoplasm interphase in certain integrin conformation(due south). The membrane proteins usually have a tryptophan or a tyrosine at the ends of the TM segments where they tin serve every bit anchors by interacting both with the fatty acid chains and the carbonyl group of the phospholipids via hydrophobic and hydrogen bonds, respectively (

). The bones residue (e.g. Lys⁷⁵² in β₁) may serve as a flexible anchor that can collaborate via its long side chain with the negatively charged phosphate groups of phospholipids even if the α-carbon moves a short distance in or out of the membrane.

It has been suggested that 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 equally a rigid piston through the membrane (

) (Fig. 6A). Because the extracellular region immediately outside of the TM domains analyzed in this study contains a short stretch of nonpolar or weakly polar residues, such a model appeared to be possible. Still, we find that the position of the N-terminal terminate of the TM helix remains unaltered when the position of the C-concluding stop is forced to shift from Phe-Lys¹⁰²⁷ to Tyr-Lys¹⁰²² for α₅ and from Ile-His⁷⁵⁸ to Trp-Lys⁷⁵² for β₁ by replacing a leucine with lysine at positions 1023 and 753, respectively (

). Therefore, the piston model seems unlikely for the α_five and β_ane subunits. If the C-terminal end of the TM domains is induced to motility into the cytoplasm by physiological stimuli, e.g. by a poly peptide-poly peptide 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. six (B and C). Farther experiments will exist needed to test whether alterations in the orientation of the membrane-proximal region of one or both integrin subunits are linked to the agile, inactive, or ligand-stimulated conformations.

Acknowledgments

Nosotros give thanks 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 help.

REFERENCES

1. • Schwartz Yard.A.
   • Schaller Grand.D.
   • Ginsberg M.H.
   Annu. Rev. Jail cell Dev. Biol. 1995; 11 : 549-599
2. • Hynes R.O.
   Dev. Biol. 1996; 180 : 402-412
3. • Takagi J.
   • Petre B.
   • Walz T.
   • Springer T.
   Jail cell. 2002; 110 : 599
4. • Carman C.Five.
   • Springer T.A.
   Curr. Opin. Jail cell Biol. 2003; 15 : 547-556
5. • Leisner T.M.
   • Wencel-Drake J.D.
   • Wang W.
   • Lam S.C.
   J. Biol. Chem. 1999; 274 : 12945-12949
6. • Humphries M.J.
   • Symonds E.J.
   • Mould A.P.
   Curr. Opin. Struct. Biol. 2003; xiii : 236-243
7. • Calderwood D.A.
   • Zent R.
   • Grant R.
   • Rees D.J.
   • Hynes R.O.
   • Ginsberg M.H.
   J. Biol. Chem. 1999; 274 : 28071-28074
8. • Tadokoro S.
   • Shattil Southward.J.
   • Eto Thousand.
   • Tai Five.
   • Liddington R.C.
   • de Pereda J.M.
   • Ginsberg 1000.H.
   • Calderwood D.A.
   Science. 2003; 302 : 103-106
9. • Kim M.
   • Carman C.Five.
   • Springer T.A.
   Scientific discipline. 2003; 301 : 1720-1725
10. • Garcia-Alvarez B.
    • de Pereda J.1000.
    • Calderwood D.A.
    • Ulmer T.S.
    • Critchley D.
    • Campbell I.D.
    • Ginsberg M.H.
    • Liddington R.C.
    Mol. Cell. 2003; eleven : 49-58
11. • Takagi J.
    • Erickson H.P.
    • Springer T.A.
    Nat. Struct. Biol. 2001; 8 : 412-416
12. • Li R.
    • Mitra N.
    • Gratkowski H.
    • Vilaire G.
    • Litvinov R.
    • Nagasami C.
    • Weisel J.W.
    • Lear J.D.
    • DeGrado W.F.
    • Bennett J.S.
    Science. 2003; 300 : 795-798
13. • Williams Chiliad.
    • Hughes P.E.
    • O'Toole T.East.
    • Ginsberg M.H.
    Trends Prison cell Biol. 1994; iv : 109-112
14. • Adair B.D.
    • Yeager M.
    Proc. Natl. Acad. Sci. U. Due south. A. 2002; 99 : 14059-14064
15. • Armulik A.
    • Nilsson I.
    • von Heijne Yard.
    • Johansson S.
    J. Biol. Chem. 1999; 274 : 37030-37034
16. • O'Toole T.E.
    • Katagiri Y.
    • Faull R.J.
    • Peter Grand.
    • Tamura R.
    • Quaranta V.
    • Loftus J.C.
    • Shattil S.J.
    • Ginsberg M.H.
    J. Cell Biol. 1994; 124 : 1047-1059
17. • Gottschalk 1000.E.
    • Adams P.D.
    • Brunger A.T.
    • Kessler H.
    Poly peptide Sci. 2002; xi : 1800-1812
18. • Nilsson I.
    • Saaf A.
    • Whitley P.
    • Gafvelin G.
    • Waller C.
    • von Heijne G.
    J. Mol. Biol. 1998; 284 : 1165-1175
19. • Liljestrom P.
    • Garoff H.
    J. Virol. 1991; 65 : 147-154
20. • Dempski Jr., R.East.
    • Imperiali B.
    Curr. Opin. Chem. Biol. 2002; 6 : 844-850
21. • Vinogradova O.
    • Haas T.
    • Plow Due east.F.
    • Qin J.
    Proc. Natl. Acad. Sci. U. S. A. 2000; 97 : 1450-1455
22. • van der Flier A.
    • Sonnenberg A.
    Jail cell Tissue Res. 2001; 305 : 285-298
23. • DeMali Thou.A.
    • Wennerberg K.
    • Burridge K.
    Curr. Opin. Cell Biol. 2003; 15 : 572-582
24. • Armulik A.
    • Velling T.
    • Johansson Due south.
    Mol. Biol. Jail cell. 2004; ()
25. • Xiong Y.Yard.
    • Chen J.
    • Zhang L.
    J. Immunol. 2003; 171 : 1042-1050
26. • Li R.
    • Babu C.R.
    • Valentine One thousand.
    • Lear J.D.
    • Wand A.J.
    • Bennett J.Southward.
    • DeGrado W.F.
    Biochemistry. 2002; 41 : 15618-15624
27. • Argraves W.S.
    • Suzuki Due south.
    • Arai H.
    • Thompson K.
    • Pierschbacher M.D.
    • Ruoslahti Eastward.
    J. Cell Biol. 1987; 105 : 1183-1190
28. • Camper Fifty.
    • Hellman U.
    • Lundgren-Akerlund E.
    J. Biol. Chem. 1998; 273 : 20383-20389
29. • Tuckwell D.Due south.
    • Humphries M.J.
    FEBS Lett. 1997; 400 : 297-303
30. • Takada Y.
    • Hemler M.E.
    J. Prison cell Biol. 1989; 109 : 397-407
31. • Schneider D.
    • Engelman D.G.
    J. Biol. Chem. 2004; 279 : 9840-9846
32. • Killian J.A.
    • von Heijne Thousand.
    Trends Biochem. Sci. 2000; 25 : 429-434
33. • Moyle M.
    • Napier Grand.A.
    • McLean J.W.
    J. Biol. Chem. 1991; 266 : 19650-19658

Article Info

Publication History

Published online: March 10, 2004

Received in revised form: March 10, 2004

Received: January 23, 2004

Identification

DOI: https://doi.org/10.1074/jbc.M400771200

Copyright

© 2004 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biological science.

User License

Creative Commons Attribution (CC BY 4.0) |

ScienceDirect

Admission this article on ScienceDirect

• View Large Image
• Download Hello-res image
• Download .PPT

Related Articles

youngfroff1956.blogspot.com

Source: https://www.jbc.org/article/S0021-9258(20)66980-0/abstract

Share this post