[Frontiers in Bioscience 7, d219-227, January 1, 2002]


Annika Armulik

Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, S-751 23, Uppsala, Sweden


1. Abstract
2. Introduction
3. Splice variants of b1
4. Splice variant b1A
5. Splice variantb1B
5. Splice variants b1C-1 and b1C-2
6. Splice variant b1D
7. Conclusions
8. Acknowledgements
9. References


The integrin b1 subfamily of adhesion receptors consists of 12 members and forms the biggest subfamily among integrins. Human integrin subunit b1 has five cytoplasmic splice variants (b1A, b1B, b1C-1, b1-C2, b1D). Even though cytoplasmic splice variants do not change the ligand-specificity of a b1 integrin, clustering of these different splice variants triggers signaling pathways that lead to a different cellular response. The main focus of this review is on the origin and specific functions of the less abundant human integrin b1 splice variants (B, C-1, C-2, D).


Integrins constitute a major group of cell-surface receptors for extracellular matrix and cell-surface proteins. The term "integrin" was introduced by R. Hynes and co-workers in 1986 and designates a cell surface receptor that connects cell cytoskeleton to the extracellular matrix (ECM). Since the publication of the full-length cDNAs of the fibronectin receptor (current name a5b) in 1986 - 1987 (1, 2), 18 a and 8 b subunits are now described in mammals. An integrin is composed of non-covalently associated transmembrane a and b subunits. The known combinations of different a and b subunits give rise to 24 different heterodimers. The integrin family can conditionally be divided into subfamilies dependent on their subunit composition and ligand specificity. The main focus of this review - integrin subunit b1 can combine with 12 a subunits and forms the biggest integrin subfamily with broad ligand specificity.

Importantly, integrins do not act only as molecular bridges linking intracellular filament systems (actin filaments and intermediate filaments) with ECM but are also important for mediating signals from ECM that regulate growth, death, differentiation, and movement of cells. Since integrins do not possess any known intrinsic kinase activity they transduce signals by spatially compartmentalizing docking and adapter proteins that link integrins to cytoplasmic kinases. Furthermore, cells themselves can dynamically regulate integrin-mediated cell adhesion. Modulation of integrin affinity by intracellular factors results in extensive conformational changes in the receptor that affect the ligand-binding interface.

The short cytoplasmic tails of integrins are absolutely required for integrin activation and signaling. Great efforts have been made to identify residues in the cytoplasmic tail of b subunits required for these specific aspects of integrin function. In particular, the cytoplasmic domains of b1, b2, and b3 have been subjects for extensive studies regarding the effects of deletions and amino acid substitutions.

Integrin signaling is complex and far from being understood. One given receptor can often bind different ligands that in turn result in activation of different signaling pathways. One should keep in mind that integrin signaling is often cell-type specific. In addition, integrins exist in several splice variants (extracellular- and intracellular) that further increases the complexity. The highly recommendable review by de Melker and Sonnenberg gives a good overview about extra- and intracellular splice variants of different integrins (3). The aim of this review is to describe some of the integrin b1 cytoplasmic tail splice variants in more detail and underline some specific aspects of splice variants b1B, b1C-1 and b1C-2 that have been largely neglected.


Integrin subunit b1 is expressed in all mammalian cells, except for mature erythrocytes. Knockout experiments in mice have shown that integrin subunit b1 is absolutely required for embryonic development (4, 5). Specific deletion of b1 integrin subunit in the nervous system (in neurons and glia cells) showed that b1 integrins are required for anchorage of glial endfeet, the remodeling of basement membranes but not essential for neuron-glia interactions and neuronal migration during corticogenesis (6). Fetal and adult blood stem cells lacking b1 integrins cannot colonize hematopoetic organs (7). In skin, b1 integrins are required for hair follicle development and the maintainace of the epidermal-dermal junction (8).

For human b1, five different cytoplasmic splice-variants are characterized, namely b1A, b1B, b1C-1, b1C-2, b1D (Figure 1) (9-14). All splice-variants of human b1 share the common N-terminal part until the sequence WDT777 that corresponds to the 3' end of exon 6 in the b1 gene (Figure 1). We know for today that the cytoplasmic splice-variants of b1 do not change the ligand specificity for a given heterodimer, but they can modulate receptor affinity towards the ligand (15, 16). Essentially nothing is known about specific intracellular signals that these variants may generate.


The splice-variant A, mostly referred to as b1 only, is very conserved at the amino acid level amongst different species from sponge to human, particularly in the transmembrane and cytoplasmic domains (17). Most review articles dealing with integrin b1 signaling and affinity regulation are covering data obtained by studying b1A and therefore b1A will not be discussed in this review. For further information, the reader is referred to several review articles covering integrin activation and signaling (18-21).


The b1B isoform was isolated from a human placenta library probed with a synthetic oligonucleotide corresponding to the cytoplasmic domain of b1A. The last 12 amino acids of b1B that are different from b1A are derived from the intronic sequence that follows immediately downstream of exon 6 (Figure 1 and 2) (9). Analysis of the nucleotide sequence of the mouse b1 gene has revealed that the mouse intronic sequence after exon 6 could potentially code for 15 amino acids (VSYETLLRAVGWFLK) that show no significant homology to human b1B, except for first three amino acids (10). Thus, the human b1B described in the literature has no orthologue in mouse; it is not known how is the situation in primates.

The b1B specific transcript has been detected at low levels in all human tissues and cell lines tested by RT-PCR, but the protein was reported to be detectable only in skin (keratinocytes) and liver (hepatocytes) (9, 22). Expression of human b1B in CHO cells showed that b1B can dimerize with a subunits and bind to a fibronectin affinity matrix in an RGD-dependent manner in the presence of Mn2+. In contrast to b1A, the b1B integrins did not localize to focal contacts when cells were plated on fibronectin (22). However, b1B can accumulate to some extent to focal contacts in a ligand-independent manner (15). Further analysis revealed that the human b1B isoform does not mediate cell spreading and activation of focal adhesion kinase (FAK) in cells plated on anti-human b1 mAb (TS2/16) (23). Furthermore, induction of ligand occupied conformation by the small GRGDS peptide of b1B integrins does not trigger activation of FAK and tyrosine phoshorylation of paxillin (15); A.Armulik, unpublished results). The cytoplasmic part common for all integrin b1 splice-variants is thus not sufficient for FAK activation. Interestingly, triggering of a ligand occupied conformation in b1B integrins results in modest tyrosine phosphorylation of CAS (A.Armulik, unpublished results).

Expression of b1B in CHO cells was reported to reduce cell spreading on fibronectin and laminin-1 but not on vitronectin (23). The attachment of cells to fibronectin and laminin-1 was only affected in clones expressing high levels of b1B (50% of that of endogenous b1A). The migration on gelatin of CHO cells expressing the b1B was similar to that of CHO cells expressing b1A when vitronectin, but not fibronectin, was used as a chemoattractant. Again, similarly to cell attachment data, higher b1B expression levels caused stronger inhibition. From these experiments it was concluded that b1B has a dominant negative effect on endogenous b1 integrins and it was suggested to be caused by the competition of b1B with endogenous b1A for available a subunits (23) and subsequently a failure of b1B to bind to extracellular ligands and activate intracellular signaling pathways. b1B expressed in the b1-deficient cell line GD25, similar to b1A, dimerizes with a5, a3 and a6 subunits (24). GD25 cells do not adhere to laminin-1 but expression of b1A in GD25 cells restored the ability of these cells to attach to laminin-1 via a6b1A (25); however, the expression of b1B subunit did not promote cell adhesion unless Mn2+ was present in the medium (24). Analysis using antibodies recognizing epitopes exposed only in the ligand-competent/occupied integrins revealed that the extracellular domain of b1B integrins possesses an inactive conformation (15, 24). The inactive ectodomain conformation could be changed to active by addition of Mn2+ or the GRGDS peptide (15, 24). The spreading and organization of actin stress fibers of GD25-b1B cells on fibronectin was found to be impaired compared to GD25 cells (24). The authors concluded that b1B has a dominant negative effect not only on b1A integrins but also on avb3/b5 integrin, since the attachment of GD25 to fibronectin is mediated via these latter integrins (25). The vitronectin substrate was not tested in the former report. In contrast to this finding (24) we found that b1B does not have a dominant-negative effect over the av integrins (15). GD-25 cells expressing human b1B were not impaired in cell attachment, spreading, and in organization of actin cytoskeleton neither on fibronectin nor on vitronectin (15). As mentioned above, expression of b1B in CHO cells also did not inhibit cell spreading on vitronectin (i.e. adhesion mediated via av integrins) (23).

The b1B integrins were found not only to be unable to mediate the assembly of fibronectin matrix but were reported to inhibit this process in CHO, GD25 and FRT cells (24, 26). Overexpression of constitutively active RhoA in FRT cells abrogated the negative effect of b1B on matrix assembly (27).

Studies on human keratinocytes, one of the few cell types that was reported to express the b1B variant at a detectable protein level, showed that overexpression of b1B in keratinocytes results in intracellular accumulation of the protein, which could be overcome by deleting the KK sequence (Figure 1) (28). b1B has been suggested to have a regulatory role of adhesion-mediated signaling. However, the modulating effects of b1B over b1A have only been observed at expression levels many-fold higher than what apparently occurs in vivo. Thus, the physiological relevance (if any) of human b1B remains to be established. The b1B specific part is generated by intron retention. Similar splice variants are found for human integrin subunit b3 (b3B) and b4 (b4E) (29, 30). So far no specific function for these splice variants has been described. Intron retention resulting from aberrant pre mRNA splicing has been described for quite a number of other proteins (e.g. CD44, periaxin, rhodopsin kinase) (31-34). In most cases, intron retention results in a truncated non-functional protein with an intron-encoded C-terminus, similar to b1B. It is most likely that human b1B represents just a splicing error occurring at low frequency rather than an obscure way to regulate cell adhesion.


The transcript for the splice variant b1C-1 was first isolated from a human erythroleukemia (HEL) cell bl-cDNA library (11), and the transcript for b1C-2 was identified using RT-PCR with total RNA from the human HL60 cell line as template (12). The b1C-1 differs from b1C-2 by six amino acids (Figure 1) that in b1C-2 are missing as a result of the utilization of a more distal 3' splice acceptor site (Figure 2) (12). Similar to b1B, the splice-variants b1C-1 and b1C-2 are only found in human and not in mouse (11, 12). Exon C is part of an Alu element and Alu elements are primate specific retrotransposable elements (12). Alignment of the genomic nucleotide sequence around exon C with the reverse complement of a consensus Alu sequence clearly demonstrates the homology (Figure 3). The calculated homology at the nucleotide level between the exon C-Alu element and a consensus Alu element is 81% and within the 116 bp exon C-region, it is as high as 91% (12).

As mentioned, Alu elements are primate specific repetitious genomic DNA sequences that belong to a group of sequences called short interspersed nucleotide elements (SINEs) (35, 36), and they are present at a copy number of approximately 500 000 per haploid human genome, making up around 5% of all human DNA. Alu elements are occasionally found to be part of coding regions of mRNA (for a review see: (37). In most of these cases, the Alu-derived sequence has been included into the transcript by a splice-mediated insertion of intronic Alu sequence. Investigation of the consensus Alu element in more detail has revealed that it contains several nucleotide regions that are similar to eukaryotic splice acceptor and donor sites. The requirement for a polypyrimidine tract is met by the presence of the reverse complement of the polyadenyl tail and the adenine-rich linker located at the end, and in the middle, of the Alu element, respectively. Both sense and antisense Alu elements can be spliced into mRNA (for review see: (37). A comparison of the Alu-exon encoded amino acid sequences demonstrates that sequences translated in the same reading frames can give rise to amino acid regions of high homology (Figure 4). Thus, to some extent, inclusion of Alu-exons can provide proteins with specific Alu-derived-domains. However, the functional significance of these domains is still unclear and most of these alternative transcripts have been identified to occur at a very low frequency compared with the normal protein variants and they are all expressed simultaneously as the normal variant, similarly to b1C-1 and b1C-2.

Analysis of the genomic region around exon C has identified an upstream branch-point sequence (CTCTAAT) and several potential AG dinucleotides (Figure 3) (12). Downstream of the polypyrimidine tract, which consists mostly of thymidines, the sequence GAGACAG follows and then the b1C-1 exon starts. Within the next 18 nucleotides, another stretch of mostly pyrimidines follows before a CCAG-sequence followed by the start of the b1C-2 exon. The splice-donor site used by both b1C-1 and b1C-2 is CAG|GTCCT and contains the important AG|GT combination at the exon-intron border. Comparing the b1C-1 and b1C-2 splice sites with the consensus splice sequence shows that they are not optimal, suggesting that this might be part of the reason why exon C is included in the mature b1-mRNA at such low frequency (12).

Using RT-PCR the amount of both b1C-1 and b1C-2 transcripts have been found to be low compared to the b1A-transcript. Nevertheless, the b1C-1 and b1C-2 transcripts have been identified by RT-PCR in a whole range of human cell lines and tissues (11, 12). At the protein level, b1C has been detected from surface biotinylated HEL cells, TNFa-stimulated HUVEC, prostate carcinomas and from benign prostate tissue (11, 38-41). In these experiments, the antibody used was a peptide antibody generated against the 23 amino acid long peptide (S805-P825) deduced from the C-terminal end of the b1C-1 sequence (Figure 4) (11). This antibody would not distinguish between the b1C-1 and b1C-2 variants, thus, when considering the equal amounts of transcripts for the two b1C-variants in many cell types, the bands seen by this antibody most probably contain both b1C-1 and b1C-2 protein. Peptide antibodies raised against two regions of the b1C cytoplasmic region have been used in immunohistochemical studies. The anti-b1C (G785-S808) would in principle be able to recognize all proteins containing a similar region encoded by an Alu-derived exon, thus the specificity of this antibody is questionable (Figure 4). Twelve of the 21 amino acids constituting the peptide used to generate the anti-b1C (S805-P825) antibody are encoded by the Alu-derived exon, thus this antibody may also recognize other proteins than b1C. Regardless of this, both antibodies have been used to demonstrate a proposed specific expression of b1C in various tissues. In particular, several studies have been conducted in order to compare the expression-pattern of b1C in normal and carcinogenic human tissues (39-41), since overexpression of b1C-1 has been shown to inhibit cell proliferation in several cell lines. Using the anti-b1C peptide antibodies, a correlation between downregulated b1C-expression and neoplasia was identified. The proposed hypothesis is that normal cells express low levels of b1C in order to not become neoplastic, while reduced b1C expression would induce rapid cell proliferation. Interestingly, the anti-b1C (G785-S808) gave better immunohistochemical staining than the antibody anti-b1C (S805-P825), the latter being generated against the more C-terminal part of b1C (Figure 3) (39). This might indicate that the epitope (or epitopes) recognized by the former antibody is part of one or several proteins containing Alu-encoded sequences.

Expression of the b1C-1 cDNA in several cell lines has demonstrated that part of the b1C-specific cytoplasmic domain has inhibitory effects on cell proliferation (38, 40-43). Using deletion-mutants expressed in CHO and mouse 10T1/2 fibroblasts the growth inhibitory effect was located within the 18 amino acid long region from b1C-1 (Q795-T812) (43). Importantly, it was also shown that expression of the b1C-1 specific-domain b1C-1 (S778-P825) when fused to the green fluorescent protein (GFP) was equally potent as the intact b1C-1 protein. Hence, the growth inhibitory effect of b1C-1 is not dependent on the localization at the plasma membrane or any other feature provided by the whole b1C-protein. Information about the direct downstream effector molecules from b1C is lacking, but a cell-cycle inhibitor p27kip1 has been identified as one of the nuclear effector molecules (40). Expression of b1C-1 in CHO cells was found to have an inhibitory effect on activation of ERK2 by fibronectin but not on activation of focal adhesion kinase (FAK) or Akt. Moreover, clustering of b1C integrins was shown to lead to the activation of the Akt pathway (44). Interestingly, even though b1C has been reported to associate with a subunits (11) it has never been shown that b1C integrins can bind a ligand. So, even if a clustering of b1C-1 by mAbs could lead to a cellular response, it is unclear whether under physiological conditions b1C-1 has any signaling properties evoked by integrin ligand binding.

Furthermore, studies on b1C splice-variants expressed in the b1-deficient GD25 cells showed that these subunits are retained in the cell and degraded rather than localized to the cell surface (45).

In conclusion: altogether there are several lines of evidence that overexpression of b1C or the b1C-specific cytoplasmic domain alone can inhibit cell proliferation but the actual mechanism for this and the molecules involved have not been identified. In addition, the immunohistochemical data ought to be considered with caution until the protein(s) recognized by the anti-b1C peptide antibodies have been identified. Like the b1B variant, the physiological role of the b1C variants is questionable.


The muscle specific integrin b1 isoform, b1D, is the only splice-variant that shares significant homology with b1A throughout the cytoplasmic tail (Figure 1). The b1D specific part (the C-terminal 24 amino acids) is encoded by exon D, which is localized between exons 6 and 7 in mouse b1 gene and between exons C and 7 in human b1 gene (Figure 1B) (13, 14). The b1D mRNA from human, mouse, rat and chicken have been sequenced and demonstrated a 100% homology at the amino acid level between the species in the b1D specific part (10, 14). In vivo, the b1D splice variant is only expressed in skeletal and cardiac muscles (not in smooth muscle), and it completely displaces b1A in terminally differentiated muscle, where it associates with a7 (46). In cardiac muscle, b1D is localized to the sarcolemma, costameres and intercalated discs, and in skeletal muscle, b1D was detected in sarcolemmas, costameres, myotendinous and neuromuscular junctions (46).

Expression of b1D in CHO cells and in GD25 cells demonstrated that b1D can localize to focal adhesions when plated on fibronectin, and clustering of b1D triggers activation of FAK and MAPK pathways (16, 46). Cells expressing the b1D isoform showed reduced spreading and migration and this occurred irrespective of the type of ECM. However, expression in C2C12, REF52 or NIH3T3 cells did not have any effect on adhesion and spreading, indicating that this might be cell type specific (47). A larger fraction of b1D than b1A was found to be in constitutively active conformation when expressed in both CHO and GD25 cells. Antibodies specific for epitopes for active ligand-binding conformation on b1 integrins recognized approximately 77-88% of b1D and only 27-44% of b1A subunit containing integrin receptors. The b1D subunit was shown to be more efficient than b1A in fibronectin matrix assembly, while b1A mediated cell migration to a greater extent than b1D. The effect on cell migration might be due to the stronger ligand binding and increased association with the actin cytoskeleton of b1D integrins compared with that of b1A and aV integrins, as shown by b1D displacement of both b1A and aV from focal contacts when cells (CHO, GD25) were plated on fibronectin (16). However, expression of b1D in NIH3T3 cells did not show any displacement of b1A from focal contacts - again this might be a cell type specific event (16).

It is interesting that despite the high degree of homology between b1A and b1D, b1D integrins display an increased affinity for fibronectin and enhanced association with the actin cytoskeleton (16). In vitro binding studies have shown that the cytoplasmic domain of b1D binds the cytoskeletal proteins talin and filamin with higher affinity than b1A (16, 48). On the other hand, more a-actinin was found to be associated with b1A than with b1D (16). The talin binding-site in the b1A cytoplasmic domain has been suggested to include the N780PXY motif (49, 50). Apparently, b1D contains additional residues that strengthen the talin binding; however, the amino acids involved are unknown.

Recently a novel intracellular protein, melusin, was reported to interact with the common region of the b1 cytoplasmic tail (51). Although in vitro binding assays showed that melusin is able to bind all tested b1 variants (A, B, D), its restricted expression to skeletal and cardiac muscle suggests that the physiological function of melusin would be through binding to integrin b1D.

Of the potential phosphorylation sites in the b1 cytoplasmic tail, the two threonines (amino acid T788 and T789) are the only residues that are not conserved between b1A and b1D. Interestingly, these threonines have been shown to be essential for b1A mediated cell attachment and fibronectin fibril formation (52). The mutant b1ATT788-9AA was shown to be in a conformation that is inactive for ligand binding. However, clustering by anti-b1 antibodies could induce phosphorylation of FAK, suggesting that the two threonines are not required for FAK activation, consistent wih the fact that b1D can activate FAK.

Expression of b1D in several different cell lines has provided contradictory findings regarding the effect of b1D expression on cell proliferation (43, 47). Belkin and Retta (47) demonstrated reduced BrdU incorporation in b1D expressing C2C12, REF52 and NIH3T3 cells, while Meredith et. al. (43) did not see any such negative effects after b1D expression in mouse 10T1/2 fibroblasts. Similar results were obtained by expression of the complete b1D molecule, or with only the cytoplasmic domain as fusion-protein with the extracellular and the transmembrane domain of IL2. In addition, expression of b1D in CHO cells, but not in C2C12 cells, gave increased MAP kinase activation (46, 47). Using the NIH3T3 cells, the b1D mediated growth arrest was identified to the late G1 phase before the beginning of the S phase, and overexpression of a constitutively active form of Ha-Ras (but not Raf-1) could abolish the growth arrest. In most pathways, Raf-1 is located downstream of Ras. However, in this case, alternative pathways must be involved. In contrast to the results from b1C, no short growth-inhibitory motif could be identified in b1D (43, 47). Instead, the only deletion mutant that did not affect the growth inhibition was a deletion of the C-terminal 6 amino acids.

Growth-arrest by expression of integrin b1D fits well with the observed onset of expression in muscle-tissues during embryogenesis where myoblasts fuse to form myotubes and stop proliferating (53, 54). In mouse embryos, b1A is the only b1 isoform expressed in skeletal muscle until embryonic day 17.5 (E17.5). After this, b1D is co-expressed with b1A until birth when b1A expression declines in skeletal muscle tissue and is restricted to the capillary walls, while b1D expression is located to the sarcolemma of the muscle cells. In cardiac muscle the situation is different. Brancaccio et al. (53) has reported that b1D is expressed already at embryonic day 11, while van der Flier et al. (54) claim that the onset of b1D expression is around the time of birth. In another report it was shown that expression of b1D can be detected at embryonic day 12 (in rat) in heart and prenatal expression of b1D was found to be <20% of that in adult ventricle (55). In any case, the b1A expression in cardiac muscle is negligible a few days after birth. The switch from expression of b1A to b1D also involves a change in the associated a-subunits. Undifferentiated C2C12 cells b1A in association with a3A, a5, a7B, and aV, while differentiated cells expressed b1D in association with a5, a7A (some a7B), and aV (54). In both skeletal and cardiac muscle, the onset of b1D parallels the start of a7 expression (53, 54, 56). Both a7A and a7B are expressed in skeletal muscle, while in cardiac muscle only a7B is expressed.

It has been suggested that replacement of the b1A isoform in muscles with b1D might be necessary to strengthen the cytoskeletal-matrix link in muscle cells (16). However, the lack of b1D isoform in transgenic mice (due to the exon D knockout) did not affect muscle formation and did not cause muscular degeneration. However, these mice showed some indications a mild ventricular dysfunction (57), and indeed, the requirement of b1D in the hyperthrophic growth of the cardiomyocytes was recently demonstrated (58). Little is known about the signaling pathways involved but there are indications that FAK is involved (55). In the converse situation, mice which express only the b1D variant (knock-in) were not viable and died in uteri because of a wide range of developmental defects (57). Embryonic b1D knock-in stem cells displayed reduced migratory activity. Expression levels of the b1D subunit were reduced when compared to b1A in wt embryonic stem cells; this could indicate that, when associated with other subunits than muscle-specific a7, the b1D protein is less stable (57).

Thus, even though b1A and b1D only differ by 13 amino acids, this difference is enough to have drastic effects on the function of the integrin, and further studies are needed to determine the critical residues and the signaling pathways involved.


Integrin signaling is complex and far from being understood. Cytoplasmic splice variants of integrin subunits add an additional level of complexity in integrin signaling. Even though five splice variants for human integrin subunit b1 have been described, most likely only two of them (A and D) are proteins with physiological functions. For three other splice variants (b1B, b1C-1 and b1C-2) the available data do not convincingly support the proposed view of their roles as physiological negative regulator of cell adhesion (b1B) or tumour-suppressor protein (b1C). Instead, these variants share characteristics typical for products resulting of abberant pre-mRNA splicing.


I would like to thank Dr. Svineng for rendered assistance.


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Key Words: Integrin, integrin subunit beta1, splice variant, signaling, Alu element, Review

Send correspondence to: Dr Annika Armulik, Laboratory of Genetics, Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute S-171 77, Stockholm, Sweden, Tel: +46-8-728 7271, Fax: +46-8-30-83-74 , E-mail: Annika.Armulik@cmb.ki.se