scholarly journals Assembly and function of integrin receptors is dependent on opposing alpha and beta cytoplasmic domains.

1995 ◽  
Vol 6 (8) ◽  
pp. 997-1010 ◽  
Author(s):  
R Briesewitz ◽  
A Kern ◽  
E E Marcantonio
Keyword(s):  
Cytoplasmic Domain ◽  
Beta Subunits ◽  
Focal Contact ◽  
Wild Type ◽  
Cytoplasmic Domains ◽  
Integrin Receptors ◽  
Integrin Receptor ◽  
And Function ◽  
Intracellular Domains ◽  
Receptor Assembly

The membrane proximal regions of integrin alpha and beta subunits are highly conserved in evolution. In particular, all integrin alpha subunits share the KXGFFKR sequence at the beginning of their cytoplasmic domains. Previous work has shown that this domain is important in integrin receptor assembly. Using chimeric integrin alpha and beta subunits, we show that the native cytoplasmic domains of both subunits must be present for efficient assembly. Most strikingly, chimeric alpha 1 and beta 1 subunits with reciprocally swapped intracellular domains dimerize selectively into collagen IV receptors expressed at high levels on the surface. However, these receptors, which bind ligand efficiently, are deficient in a variety of post-ligand binding events, including cytoskeletal association and induction of tyrosine phosphorylation. Furthermore, deletion of the distal alpha cytoplasmic domain in the swapped heterodimers leads to ligand-independent focal contact localization, which also occurs in wild-type subunits when the distal cytoplasmic domain is deleted. These results show that proper integrin assembly requires opposed alpha and beta cytoplasmic domains, and this opposition prevents ligand-independent focal contact localization. Our working hypothesis is that these two domains may associate during receptor assembly and provide the mechanism for integrin receptor latency.

scholarly journals The membrane-cytoplasm interface of integrin alpha subunits is critical for receptor latency.

1996 ◽  
Vol 7 (10) ◽  
pp. 1499-1509 ◽  
Author(s):  
R Briesewitz ◽  
A Kern ◽  
L B Smilenov ◽  
F S David ◽  
E E Marcantonio
Keyword(s):  
Ligand Binding ◽  
Point Mutations ◽  
Focal Contact ◽  
Amino Acid Residues ◽  
Cytoplasmic Domains ◽  
Integrin Receptors ◽  
Integrin Receptor ◽  
Relationship Of ◽  
Intracellular Domains ◽  
Human Integrin

Localization of integrin receptors to focal contact sites occurs upon ligand binding. This activity is latent, since unoccupied integrin receptors do not localize to focal contacts. Deletion analysis has revealed that the alpha cytoplasmic domains is required for the maintenance of integrin receptor latency. Our current hypothesis for the mechanism of integrin post-ligand binding events is that there is a change in relationship of alpha and beta cytoplasmic domains, which overcomes receptor latency. One possible mechanism for such a change would involve the amino acid residues at the membrane-cytoplasm interface. To test this hypothesis, we have produced point mutations in the human integrin alpha 1 subunit. These mutations had no effect on the adhesion via alpha 1 beta 1 to its ligand, collagen IV. However, receptor latency is lost in one of these mutants, leading to constitutive focal contact localization. This effect did not occur in receptors with an exchange of intracellular domains, suggesting that the mechanism of loss of latency involves a relative motion of the integrin chains. These results suggest a model in which post-ligand binding events in integrin receptors are associated with changes in the position of the alpha and beta cytoplasmic domains.

scholarly journals A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

1994 ◽  
Vol 14 (11) ◽  
pp. 7404-7413 ◽  
Author(s):  
S Takaki ◽  
H Kanazawa ◽  
M Shiiba ◽  
K Takatsu
Keyword(s):  
Signal Transduction ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Beta Chain ◽  
Cytoplasmic Domains ◽  
Interleukin 5 ◽  
Alpha Chain ◽  
Gm Csf ◽  
Response Expression ◽  
And Function

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

Cardiac expression of a gain-of-function α5-integrin results in perinatal lethality

2001 ◽  
Vol 280 (1) ◽  
pp. H361-H367 ◽  
Author(s):  
Maria L. Valencik ◽  
John A. McDonald
Keyword(s):  
Cardiac Development ◽  
Wild Type ◽  
Integrin Receptors ◽  
Gain Of Function ◽  
Transgenic Expression ◽  
Physiological Regulation ◽  
Myocyte Differentiation ◽  
Perinatal Lethality ◽  
And Function

Communication between the extracellular matrix and the intracellular signal transduction and cytoskeletal system is mediated by integrin receptors. α5β1-Integrin and its cognate ligand fibronectin are essential in development of mesodermal structures, myocyte differentiation, and normal cardiac development. To begin to explore the potential roles of α5β1-integrin specifically in cardiomyocytes, we used a transgenic expression strategy. We overexpressed two forms of the human α5-integrin in cardiomyocytes: the full-length wild-type α5-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated α5-1-integrin. Overexpression of the wild-type α5-integrin has no detectable adverse effects in the mouse, whereas expression of α5-1-integrin caused electrocardiographic abnormalities, fibrotic changes in the ventricle, and perinatal lethality. Thus physiological regulation of integrin function appears essential for maintenance of normal cardiomyocyte structure and function. This strengthens the role of inside-out signaling in regulation of integrins in vivo and suggests that integrins and associated signaling molecules are important in cardiomyocyte function.

scholarly journals Ligand-dependent and -independent integrin focal contact localization: the role of the alpha chain cytoplasmic domain.

1993 ◽  
Vol 4 (6) ◽  
pp. 593-604 ◽  
Author(s):  
R Briesewitz ◽  
A Kern ◽  
E E Marcantonio
Keyword(s):  
Cell Lines ◽  
Cellular Localization ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Striking Difference ◽  
Focal Contact ◽  
Integrin Receptors ◽  
Independent Manner ◽  
Contact Sites ◽  
Focal Contacts

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta 1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha 1 wild-type subunit (HA1 cells) or the cytoplasmic domain deleted alpha 1 subunit (CYT cells). Both cell lines express similar levels of the human alpha 1 subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha 1 beta 1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha 5 beta 1 but not for alpha 1 beta 1, there is a striking difference in the cellular localization of alpha 1 beta 1. The HA1 cells show only alpha 5 in focal contacts, without alpha 1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha 1 appears in focal contacts along with the alpha 5/beta 1. Thus, there is both ligand-dependent (alpha 5/beta 1) and ligand-independent (alpha 1/beta 1) focal contact localization in these cells. The truncated alpha 1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha 1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.

scholarly journals Characterization of beta pat-3 heterodimers, a family of essential integrin receptors in C. elegans.

1995 ◽  
Vol 129 (4) ◽  
pp. 1127-1141 ◽  
Author(s):  
S N Gettner ◽  
C Kenyon ◽  
L F Reichardt
Keyword(s):  
Caenorhabditis Elegans ◽  
Cytoplasmic Domain ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Integrin Receptors ◽  
Diverse Range ◽  
C Elegans ◽  
Alpha Subunits ◽  
Sds Page

Members of the integrin family of cell surface receptors have been shown to mediate a diverse range of cellular functions that require cell-cell or cell-extracellular matrix interactions. We have initiated the characterization of integrin receptors from the nematode Caenorhabditis elegans, an organism in which genetics can be used to study integrin function with single cell resolution. Here we report the cloning of an integrin beta subunit from C. elegans which is shown to rescue the embryonic lethal mutation pat-3(rh54) and is thus named beta pat-3. Analysis of the deduced amino acid sequence revealed that beta pat-3 is more similar to Drosophila integrin beta PS and to vertebrate integrin beta 1 than to other integrin beta subunits. Regions of highest homology are in the RGD-binding region and in the cytoplasmic domain. In addition, the 56 cysteines present in the majority of integrin beta subunits are conserved. A major transcript of approximately 3 kilo-base pairs was detected by RNA blot analysis. Immunoblot analysis using a polyclonal antiserum against the cytoplasmic domain showed that beta pat-3 migrates in SDS-PAGE with apparent M(r) of 109 k and 120 k under nonreducing and reducing conditions, respectively. At least nine protein bands with relative molecular weights in the range observed for known integrin alpha subunits coprecipitate with beta pat-3, and at least three of these bands migrate in SDS-PAGE with increased mobility when reduced. This behavior has been observed for a majority of integrin alpha subunits. Immunoprecipitations of beta pat-3 from developmentally staged populations of C. elegans showed that the expression of several of these bands changes during development. The monoclonal antibody MH25, which has been postulated to recognize the transmembrane component of the muscle dense body structure a (Francis, G. R., and R. H. Waterston. 1985. Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization. J. Cell Biol. 101:1532-1549), was shown to recognize beta pat-3. Finally, immunocytochemical analysis revealed that beta pat-3 is expressed in the embryo and in many cell types postembryonically, including muscle, somatic gonad, and coelomocytes, suggesting multiple roles for integrin heterodimers containing this beta subunit in the developing animal.

scholarly journals Mapping of the functional determinants of the integrin beta 1 cytoplasmic domain by site-directed mutagenesis.

1990 ◽  
Vol 1 (8) ◽  
pp. 597-604 ◽  
Author(s):  
E E Marcantonio ◽  
J L Guan ◽  
J E Trevithick ◽  
R O Hynes
Keyword(s):  
Consensus Sequence ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Focal Contact ◽  
Wild Type ◽  
3T3 Cells ◽  
Transmembrane Region ◽  
High Level Expression ◽  
Focal Contacts ◽  
High Level

We describe here the expression of deletion mutants of the cytoplasmic domain of the avian integrin beta 1 subunit. These mutants, which contain termination codons at positions 767, 776, 791, and 800, were transfected into mouse 3T3 cells to determine which sequences were essential for localization of integrins into focal contact sites. In all cases, high-level expression of the truncated avian integrins was obtained. Heterodimers were formed between the exogenous truncated avian beta 1 subunits and endogenous mouse alpha subunits, and these heterodimers were efficiently exported to the cell surface. The longest truncated beta 1 subunit tested, which is only four amino acids shorter than the wild type, does localize to focal contacts. In contrast, beta 1 subunits with moderately long truncations of the cytoplasmic domain failed to localize to focal contacts, including one which contains the consensus sequence for tyrosine phosphorylation. Surprisingly, a mutant subunit in which the bulk of the cytoplasmic domain was missing (but the segment nearest the membrane including the dibasic residues (RR) remained) did localize weakly to focal contacts. These results implicate the peptide segment nearest to the transmembrane region in focal contact localization. In addition, mutant subunits that included this segment together with a larger portion of the cytoplasmic domain did not localize as well as the shorter form, suggesting that these cytoplasmic domain segments are defective, presumably because of abnormal folding.

scholarly journals Modulation of Integrin Activity is Vital for Morphogenesis

1998 ◽  
Vol 141 (4) ◽  
pp. 1073-1081 ◽  
Author(s):  
Maria D. Martin-Bermudo ◽  
Olga M. Dunin-Borkowski ◽  
Nicholas H. Brown
Keyword(s):  
Cytoplasmic Domain ◽  
Wild Type ◽  
Integrin Receptors ◽  
Adhesive Properties ◽  
Α Subunit ◽  
Integrin Αiibβ3 ◽  
Visceral Mesoderm ◽  
The Right ◽  
Mutant Forms ◽  
Inside Out

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the α subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (αPS2βPS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type αPS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the αPS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the αPS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin αIIbβ3.

scholarly journals Requirements for the cytoplasmic domain of the alphaPS1, alphaPS2 and betaPS integrin subunits during Drosophila development

Development ◽  
1998 ◽  
Vol 125 (4) ◽  
pp. 701-711
Author(s):  
X. Li ◽  
M.W. Graner ◽  
E.L. Williams ◽  
C.E. Roote ◽  
T.A. Bunch ◽  
...  
Keyword(s):  
Temperature Shift ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Amino Acid Residues ◽  
Cytoplasmic Domains ◽  
Mutant Proteins ◽  
Adhesive Interactions ◽  
And Function ◽  
The Relationship ◽  
Tail Function

The integrins are a family of transmembrane heterodimeric proteins that mediate adhesive interactions and participate in signaling across the plasma membrane. In this study we examine the functional significance of the cytoplasmic domains of the alphaPS1, alphaPS2 and betaPS subunits of the Drosophila Position Specific (PS) integrin family by analyzing the relationship between cytoplasmic domain structure and function in the context of a developing organism. By examining the ability of ssPS molecules lacking the cytoplasmic domain to rescue embryonic abnormalities associated with PS integrin loss, we find that although many embryonic events require the betaPS cytoplasmic domain, this portion of the molecule is not required for at least two processes requiring PS integrins: formation of midgut constrictions and maintaining germband integrity. Furthermore, our studies demonstrate that mutant proteins affecting four highly conserved amino acid residues in the cytoplasmic tail function with different efficiencies during embryonic development, suggesting that interaction of PS integrins with cytoplasmic ligands is developmentally modulated during embryogenesis. We have also examined the ability of alphaPS1 and alphaPS2 to function without their cytoplasmic domains. By analyzing the ability of transgenes producing truncated alphaPS molecules to rescue abnormalities associated with integrin loss, we find that the cytoplasmic tail of alphaPS2 is essential for both embryonic and postembryonic processes, while this portion of alphaPS1 is not required for function in the wing and in the retina. Furthermore, temperature-shift experiments suggest roles for the alphaPS2 cytoplasmic domain in signaling events occurring in the developing wing.

scholarly journals Interchangeable alpha chain cytoplasmic domains play a positive role in control of cell adhesion mediated by VLA-4, a beta 1 integrin.

1993 ◽  
Vol 178 (2) ◽  
pp. 649-660 ◽  
Author(s):  
P D Kassner ◽  
M E Hemler
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Deletion Mutant ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Cytoplasmic Domains ◽  
Positive Role ◽  
Alpha Chain ◽  
Poor Adhesion

Integrins can exist in a range of functional states, depending on the cell type and its state of activation. Although the mechanism that controls activity is unknown, it has been suggested that for some integrins, alpha chain cytoplasmic domains may exert either a negative effect or no effect on adhesion function. To address this issue for VLA-4 (an alpha 4 beta 1 heterodimer), we constructed an alpha 4 cytoplasmic deletion mutant and chimeric alpha chains composed of the extracellular domains of alpha 4 and the cytoplasmic domains of alpha 2, alpha 4, or alpha 5. Upon stable transfection of wild-type alpha 4, VLA-4 heterodimer was obtained that mediated (a) poor adhesion to CS1 peptide, fibronectin, or vascular cell adhesion molecule 1 (VCAM-1) (in K562 cells); (b) poor adhesion to CS1 peptide but moderate adhesion to VCAM-1 (in MIP101 cells); and (c) moderate adhesion to both CS1 peptide and VCAM-1 (in PMWK cells). Chimeric alpha 4 constructs and wild-type alpha 4 yielded similar results in these cell lines. In contrast, truncation of the alpha 4 cytoplasmic domain (after the conserved GFFKR motif) caused an almost complete loss of adhesive activity in all three cell lines. Thus, several interchangeable alpha chain cytoplasmic domains play a fundamentally positive role in determining the state of constitutive activity for VLA-4. The alpha chain cytoplasmic domain is also required for agonist-stimulated adhesion, since phorbol ester stimulated the cell adhesion mediated by wild-type and chimeric alpha chains, but not by the cytoplasmic deletion mutant. The inactivity of both wild-type VLA-4 (in K562 cells), and truncated VLA-4 (in all three cell lines) was overcome by the addition of a stimulatory anti-beta 1 monoclonal antibody. Thus, the alpha cytoplasmic domain-dependent cellular mechanism controlling both constitutive and agonist-stimulated VLA-4 activity could be bypassed by external manipulation of the integrin.

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction

1994 ◽  
Vol 14 (11) ◽  
pp. 7404-7413
Author(s):  
S Takaki ◽  
H Kanazawa ◽  
M Shiiba ◽  
K Takatsu
Keyword(s):  
Signal Transduction ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Beta Chain ◽  
Cytoplasmic Domains ◽  
Interleukin 5 ◽  
Alpha Chain ◽  
Gm Csf ◽  
Response Expression ◽  
And Function

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

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