©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Integrin Affinity States through an NP XY Motif in the Subunit Cytoplasmic Domain (*)

Timothy E. O'Toole (§) , Jari Ylanne (1), Brian M. Culley

From the (1) Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037 Department of Biochemistry, University of Helsinki, 00014 Helsinki, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The ligand binding affinities of the integrins are regulated through their cytoplasmic domains. To identify specific residues that are involved in this process, we have generated mutants in the and tails and coexpressed them in Chinese hamster ovary cells with constitutively active subunits. These subunits are chimera of extracellular and transmembrane joined to the cytoplasmic domains of , , or and confer an energy-dependent high affinity state when expressed in Chinese hamster ovary cells. The affinity state of these transfectants was determined by analyzing the binding of PAC1, an antibody that specifically recognizes the activated form of the reporter group, extracellualar . We have identified point mutants in several areas of the tails, which result in a reduced ability to bind ligand. Complete abolition of PAC1 binding was obtained with mutants in an NP XY motif found in many integrin subunits and implicated in the internalization of other cell surface receptors. Similar effects on PAC1 binding were observed whether coexpression was with chimera containing , , or cytoplasmic sequences. These studies identify a novel role for the NP XY motif in the regulation of integrin binding affinity.


INTRODUCTION

The regulated adhesion of cells to each other or to elements in the extracellular environment is crucial in normal physiology. The integrins, a widely distributed family of , heterodimers (1) , mediate many of these events. Members of this family are characterized by an ability to dynamically regulate their ligand binding affinity. In a process termed ``activation'' or ``inside-out signaling,'' integrins change extracellular conformation and alter ligand binding affinity and cellular adhesion (1) . Following ligand binding, events are triggered inside the cell. ``Outside-in signaling'' events include changes in cell shape, integrin localization, intracellular pH, induction of protein phosphorylation, and gene transcription (1) .

The cytoplasmic sequences of the integrins appear to be involved in these bi-directional signaling events. Indeed, several functions have been associated with the subunit cytoplasmic domain. For instance, these sequences contain sufficient information for specifying localization to focal adhesions (2, 3) , are required for cellular adhesion (4, 5, 6) , and are capable of associating with the cytoskeletal proteins -actinin (7) and talin (8) . Independent expression of tails results in an inhibition of endogenous integrin function in matrix assembly, cell spreading, and cell migration (2, 9) . In addition, cytoplasmic domains have copies of an internalization sequence, NP XY (10) , and several residues that are potential phosphorylation sites. The functional properties of subunit cytoplasmic sequences are less well defined. Nevertheless, these domains limit the cellular localization of integrins (11, 12, 13) and play a role in cellular adhesion, migration, and collagen gel contraction (14, 15, 16, 17, 18, 19) .

Mounting evidence implicates a role for both and cytoplasmic domains in inside-out signaling and the maintenance of high affinity binding. The identity of the tail determines the affinity state of recombinant integrins expressed in heterologous cells (20, 21) , while deletion of these sequences disrupts cellular adhesion (14, 15, 16, 17, 19) . On the other hand, a point mutation in the tail has been associated with an activation defect of both in vivo (22) and in a recombinant system (20) . Furthermore, independent coexpression of the , but not the , cytoplasmic domain can abolish ligand binding by constitutively active, transfected integrins (23) .

Little is known regarding specific or cytoplasmic residues required for high affinity binding or how these relate to other functional areas in these domains. To map these sequences in the subunit, we have mutagenized the and tails, joined them onto extracellular and transmembrane , and coexpressed these variants with cytoplasmic chimeras that confer constitutive ligand binding properties (20) . The affinity state of these transfectants was determined by analyzing their ability to bind PAC1 (24) , a monoclonal antibody that specifically recognizes the active conformation of the reporter group, . Our results have identified several residues whose substitution reduces constitutive ligand binding. Complete abolition of PAC1 binding was observed with variants in an NP XY sequence. This role in affinity modulation identifies a novel function for this motif. Furthermore, these studies discount a role for phosphorylation of integrin tails in the regulation of binding affinity and suggest cytoplasmic splice variants which lack these sequence elements may be unresponsive to normal inside-out signaling pathways.


MATERIALS AND METHODS

Antibodies and Reagents

The purification and characterization of several antibodies (D57, anti-LIBS6, PAC1) has been described elsewhere (20, 24, 25) , while a polyclonal antibody against was generated by previously described methods (26) . The antibody D57 was biotinylated with biotin- N-hydroxysuccinimide (Sigma) according to manufacturer's directions. A peptide-mimetic compound (Ro43-5054), which specifically blocks binding to , was a generous gift from Beat Steiner (F. Hoffman LaRoche, Basel, Switzerland). Oligonucleotides were synthesized on a model 391 DNA synthesizer (Applied Biosystems Inc.). Restriction endonucleases, TaqI polymerase, and other enzymes were from Boehringer Mannheim.

cDNA Constructs

The generation of several wild type (), chimeric (, , , ), or mutant (724, S752P) integrin constructs has been described previously (20, 21, 27) . Additional chimera were made by exchanging mutant cytoplasmic domains for those from wild type . Sequences encoding the mutants S790M, T793V/T794V, and N797I were recovered by HindIII digestion of the appropriate pRneo constructs (28) and ligated into HindIII-cut pCDM8 (Invitrogen, San Diego, CA). These vectors were then digested with BspHI and DraIII, and the fragment containing cytoplasmic sequences was ligated to a corresponding BspHI- DraIII fragment from CD3a (27) containing transmembrane and extracellular domains. Cytoplasmic sequences encoding the variants F771L, E774V, N785I, Y788A, YTRF, S790D, Y800F, and Y800A were first amplified by the polymerase chain reaction with the 5` oligonucleotide: GTAAAATCACTGCAGTTTGCCCTA, and the 3` oligonucleotide: TTGATTTGGAAGCTTCTGATGATC. Amplified fragments were digested with HindIII and PstI and ligated into HindIII- and PstI-digested pCDM8. These constructs were then digested with BspHI and DraIII and ligated as above to a corresponding BspHI- DraIII fragment from CD3a. The Y788F variant of was generated in a two-step polymerase chain reaction strategy. Overlapping fragments containing the desired amino acid substitution were first generated in two amplifications on wild type sequences using the oligonucleotide pairs: GGTGAAAATCCTATTTTTAAGAGTGCCG-GTAAGGTTCCTTCACAAAGAT and CGGCACTCTTAAAAATAGGATTTTCACC-ATACCTGCAACCGTTACTGCC. The amplified fragments were combined, denatured at 85 °C for 10 min, and allowed to cool to room temp. The ends were then filled in with Sequenase (29) , and double-stranded fragments amplified with the oligonucleotides: GTAAGGTTCCTTCACAAAGAT and ATACCTGCAACCGTTACTGCC. Amplified products were digested with AflII and XhoI and inserted into the AflII- XhoI-digested chimera, thereby replacing wild type cytoplasmic sequences with the mutant sequence. The cytoplasmic variants F727A/F730L/E733V, Y747A, S752A, and Y759A were generated in a two-step polymerase chain reaction strategy as above for the variant Y788F. The final amplified product was digested with MluI and XhoI and then ligated into pCDM8. These constructs were then linearized with MluI and ligated to an MluI fragment from CD3a containing the remainder of coding sequences. The B construct was made by polymerase chain reaction amplification with the oligonucleotides: CGAAAAGAATTCGCTAAATTTGAGGAAGAACGCGCCAGAGCAAAATGGGACACCAGTAAGAGAC and AGAGTCCCCGGGTCAGACCAATGACTTTAGAAAACGCCCAGCCCCGTCTCTTACTGTGC. The amplified product was digested with EcoRI and SmaI and subcloned into CD3a cut with EcoRI and SmaI. Finally, this construct was linearized with EcoRI and ligated to an EcoRI fragment from containing its extracellular and transmembrane sequences. All constructs were identified by restriction digestion, purified by CsCl centrifugation, and verified by DNA sequencing before transfection.

Cell Culture and Transfection

CHO()cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (DMEM, BioWhittaker Inc., Walkersville, MD) supplemented with 10% fetal bovine serum (BioWhittaker Inc.), 1% L-glutamine (Sigma), 1% penicillin and streptomycin (Sigma), and 1% nonessential amino acids (Sigma).

Integrin cDNA constructs were expressed in CHO cells by liposome-mediated transfection. Twenty-four hours before transfection, CHO cells were plated at a density of 10cells/100-cm dish. A total of 2 µg of each and construct and 20 µl of Lipofectamine (Life Technologies, Inc.) reagent were incubated at room temperature for 10 min in 180 µl of unsupplemented DMEM. Unsupplemented DMEM (3.8 ml) was then added and the DNA-liposome complexes overlaid onto the cells. The cells were incubated for 6 h at 37 °C, washed with phosphate-buffered saline, and then incubated at 37 °C with complete medium. Medium was changed after 24 h and the cells harvested and analyzed at 48 h.

Immunoprecipitation

Transfectants were surface-labeled with I by the lactoperoxidase-glucose oxidase method (30) and then solubilized in lysis buffer (10 m M Hepes (pH 7.5), 0.15 M NaCl, 50 m M octyl glucoside, 1 m M CaCl, 1 m M MgCl, 1 m M phenylmethylsulfonyl fluoride, 0.1 m M leupeptin, and 10 m M N-ethylmaleimide). Cell extracts were immunoprecipitated overnight with preimmune serum or with a polyclonal antiserum directed against and then incubated with protein A-Sepharose CL-4B for 1 h at room temperature. The Sepharose beads were washed extensively in lysis buffer, resuspended in sample buffer, and boiled for 5 min. After centrifugation, immunoreactive proteins were resolved on non-reducing, 7.5% acrylamide gels, the gels dried, and bands visualized by autoradiography.

Flow Cytometry

Integrin affinity state was determined by a two color flow cytometry assay. Transient transfectants were harvested in Tyrode's buffer (31) containing 0.1 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington) and 3.5 m M EDTA. After a 5-min incubation at room temperature, the cells were diluted with Tyrode's buffer containing 0.1% soybean trypsin inhibitor (Sigma) and 10% bovine serum albumin (Sigma), collected by centrifugation at 1200 rpm for 5 min, and washed once in Tyrode's buffer containing 10% bovine serum albumin. The cells were resuspended in Tyrode's and 5 10analyzed for PAC1 binding. Briefly, the cells were incubated in a final volume of 50 µl with a 1:25 dilution of PAC1 ascites in the presence or absence of 1 µ M Ro43-5054. After 30 min at room temperature, the cells were diluted to 0.5 ml with Tyrode's buffer, pelletted, and resuspended in 50 µl of Tyrode's buffer containing a biotinylated, -specific antibody, D57. After 30 min on ice, the cells were diluted to 0.5 ml with Tyrode's buffer, pelletted, and resuspended in 50 µl of Tyrode's buffer containing 4% phycoerythrin-conjugated strepavidin (Molecular Probes Inc., Junction City, OR) and 10% FITC-conjugated goat anti-mouse IgM (Tago, Burlingame, CA). After 30 min on ice, the samples were diluted to 0.5 ml with Tyrode's buffer and analyzed by flow cytometry on a FACScan (Becton Dickinson, San Jose, CA).

PAC1 binding (FITC staining) was analyzed only on those gated cells positive for surface expression of (phycoerythrin staining). Histograms of FITC staining in the presence or absence of a competitive inhibitor of PAC1 binding, Ro43-5054, were compared. High affinity integrins were identified by a rightward shift of the histogram in the absence of Ro43-5054. PAC1 binding was also analyzed after incubation with 6 µ M anti-LIBS6. This antibody induces the high affinity state of and reports optimum ligand binding (25, 32) . As a quantitative measure of affinity state we have expressed the data as an activation index for each , pair. The activation index was defined as: 100 ( M - M/ M- M), where M = median fluorescence intensity of PAC1 binding, M= median fluorescence intensity of PAC1 binding in the presence of Ro43-5054, M= median fluorescence intensity of PAC1 binding in the presence of anti-LIBS6, and M= median fluorescence intensity of PAC1 binding in the presence of anti-LIBS6 and Ro43-5054.


RESULTS

Cytoplasmic Mutation Does Not Disrupt Surface Expression

To identify residues in cytoplasmic domains that are involved in the regulation of integrin affinity states, we have generated point mutations in these sequences and coexpressed these variants in CHO cells with chimeras. These subunit chimeras consist of the extracellular and transmembrane domains of joined to the cytoplasmic sequences of , A, or B. In contrast to wild type , these chimeras conferred an energy-dependent, constitutive high affinity state when coexpressed with constructs encoding the wild type or cytoplasmic domains (20) . Thus, the affinity state of the extracellular reporter group, , was determined by the identity of its cytoplasmic sequences.

Point mutations were first generated in the cytoplasmic sequences of (Fig. 1), a natural partner for the and cytoplasmic domains. Areas targeted included several hydroxylated residues which represent potential phosphorylation sites, the two NP XY internalization motifs, and residues in the putative -actinin binding site. Many of these residues have also been implicated in the recruitment of to focal adhesions (28) . These variant cytoplasmic sequences were first joined to the extracellular and transmembrane domains of generating subunit chimeras. To determine if cytoplasmic mutations affected integrin expression, cells cotransfected with these chimeras and (a chimera consisting of extracellular and transmembrane joined to cytoplasmic ) were analyzed for surface expression by flow cytometry. Constructs encoding the wild type and variant forms of the cytoplasmic domain demonstrated comparable levels of surface expression (Fig. 2, panels A, D, and G) when stained with an anti--specific monoclonal antibody. Indeed, all of the variants utilized in this study were well expressed. In addition, no differences between wild type or mutant expression were observed when cotransfection was with the A or B chimeras. Heterodimer formation and surface expression of variants was also confirmed by the immunoprecipitation of iodinated transfectants (data not shown). Thus, the point mutations we have examined do not disrupt normal subunit association or cell surface expression.

Mutations in the Cytoplasmic Domain Abolish Inside-out Signaling

To examine the functional effects of cytoplasmic mutation on ligand binding, transfectants were analyzed for their ability to bind PAC1 by flow cytometry (Fig. 2). Only those cells positive for surface expression (denoted by M1 in Fig. 2) were gated and analyzed for PAC1 binding. Like cells transfected with wild type ( panel B), those expressing the Y788F variant bound PAC1 constitutively ( panel H). Binding was specific since it could be blocked with a ligand-mimetic compound, Ro43-5054. In contrast, cells transfected with the Y788A variant failed to bind PAC1 ( panel E). Binding in the latter case was observed only in the presence of an activating antibody, anti-LIBS 6.


Figure 2: Representative FACS histograms. Cells transfected with the and chimera listed on the left were stained and subjected to flow cytometry as described under ``Materials and Methods.'' Phycoerythrin staining, indicative of antibody D57 binding and integrin surface expression levels, is illustrated in panels A, D, and G. Note that each heterodimer was expressed at similar levels. Only those cells positive for surface expression (denoted in each histogram by M1) were gated and analyzed for FITC staining, indicative of PAC1 binding ( panels B, C, E, F, H, and I). Histograms of PAC1 binding in the absence of inhibitor ( solid line) were superimposed upon those analyzed in the presence of 1 µ M Ro43-5054 ( dotted line). A rightward shift is indicative of an active integrin. Histograms of PAC1 binding done in the presence of 6 µ M anti-LIBS6 are shown in the last column ( panels C, F, and I). All histograms represent an acquisition of 10events. Coexpression with the and Y788F constructs result in a constitutively active integrin ( panels B and H), while expression of Y788A results in an inactive integrin ( panel E).



To numerically express PAC1 binding and affinity state, we have determined an activation index (AI) for each , combination as described under ``Materials and Methods.'' The AI values were determined for each of the tail variants when cotransfected with the chimera and compared to the wild type sequence (Fig. 3 A). When analyzed in this way, the group of variants fell into three functional categories: those that conferred near wild type levels (70-100%) of PAC1 binding (Y788F, Y800F), those that result in reduced (10-70%) binding (F771L, E774V, S790M, S790D, T793V/T794V, N797I, Y800A), and those that abolished (<10%) binding (N785I, Y788A, YTRF). To determine if these effects were specific for the cytoplasmic tail, the variants were also transfected with other constructs. Similar effects on PAC1 binding were observed when coexpression was with chimeras containing the cytoplasmic sequences from the (Fig. 3 B) and (data not shown) subunits. These results suggest that mutations at several sites within the cytoplasmic domain reduce energy-dependent ligand binding.


Figure 3: Activation indices of cytoplasmic variants. As outlined under ``Materials and Methods'' and ``Results,'' we have determined an activation index for each variant after cotransfection with chimera containing ( A) or ( B) cytoplasmic sequences. Values for cotransfection with the wild type chimera () are depicted at the bottom of each graph. The lowest activation indices (least PAC1 binding) were those obtained by transfection with the N785I, Y788A, and YTRF variants. A negative activation index results when the mean fluorescence intensity of PAC1 binding in the presence of inhibitor is greater than that value in the absence of inhibitor.



Mutations in the Cytoplasmic Domain Abolish Inside-out Signaling

The and cytoplasmic domains are approximately 60% identical and contain conservative amino acid substitutions at several other positions. This high degree of conservation suggests the possibility that analogous regions in both tails have functional importance. To look at this possibility, we have generated point mutations in the tail at sites corresponding to those residues in which affect its binding function (Fig. 1). Like those point mutations, cytoplasmic variants of do not disrupt normal patterns of , subunit association or cell surface expression (data not shown). With respect to functional properties, transfectants expressing a cytoplasmic truncation (724) or the F727A/F730L/E733V, Y747A, and S752P, variants demonstrate a greatly reduced (<10%) ability to bind PAC1 when coexpressed with the chimera (Fig. 4 A). Although still reduced relative to wild type , the Y759A and S752A variants exhibit a somewhat greater AI. Similar effects on PAC1 binding were observed when these variants were coexpressed with the (Fig. 4 B) or A (data not shown) chimeras. Thus analogous residues in the and tails affect ligand binding affinity. Finally, we have generated a construct (B) encoding an alternately spliced form of the cytoplasmic domain (33) . This variant eliminates 8 of 10 hydroxylated residues and the NP XY motifs in this tail. Coexpression of this construct with the constitutively active chimera also resulted in a low affinity receptor whose binding function was only elicited with anti-LIBS6 (Fig. 5).


Figure 1: Wild type and variant cytoplasmic sequences. Illustrated at top of figure is a schematic of the integrin chimeras used. The chimeras consist of the extracellular and transmembrane domains of joined to the cytoplasmic domains of , , or . Also represented are the wild type subunit and a chimera of extracellular and transmembrane joined to cytoplasmic . Listed below, in single-letter code, are the cytoplasmic sequences of (beginning with Lys) and the chimera. sequences begin with the underlined histidine. The position of amino acid substitutions or a termination codon ( asterisk) in these domains are illustrated. Dashes represent unchanged amino acids, while mutant name is listed on the right.




Figure 4: Activation indices of cytoplasmic variants. As outlined under ``Materials and Methods'' and ``Results,'' we have determined an activation index for each variant after cotransfection with the ( A) or the ( B) chimera. Values for cotransfection with wild type are depicted at the bottom of each graph. A negative activation index results when the mean fluorescence intensity of PAC1 binding in the presence of inhibitor is greater than that value in the absence of inhibitor.




DISCUSSION

The results described above: 1) identify a distinct set of cytoplasmic residues required for inside-out signaling (Fig. 6), 2) define a functional role for the NP XY motif in this is process, 3) argue against the hypothesis that the phosphorylation of integrin tails is required, and 4) suggest cytoplasmic splice variants are not responsive to normal inside-out signaling mechansims.

The most significant reductions in constitutive PAC1 binding were achieved by substitutions of the cytoplasmic residues Asnand Tyr. These residues encompass an NPIY sequence of this domain, a conserved motif (NP XY) found in many subunit cytoplasmic domains. Indeed, the (34) , (35) , (36) , (37) , (38) , and (39) cytoplasmic sequences typically contain one of these motifs within 30 residues of the transmembrane domain (Fig. 6). In addition, the and tails also have a more distal NP XY/F sequence, while other subunits contain a distal N XXY/F motif. The NP XY motif has been identified in the cytoplasmic sequences of several cell surface receptors (10) . It functions as an internalization sequence for the LDL receptor (10) and is essential for cellular transformation by the polyomavirus middle T antigen (40) . Functional studies suggest that the Asn and Pro residues of this motif are invariant. Substitution of either in the LDL receptor results in a significant reduction of coated pit-mediated endocytosis (10) . While internalization was maintained when the terminal Tyr of this motif was substituted with another aromatic residue (Phe, Trp), substitution with a non-aromatic residue resulted in decreased LDL receptor internalization (41) . These functional studies are similar to ours which demonstrated that substitution of Asncompletely abolished PAC1 binding. Furthermore, we have observed a complete loss of PAC1 binding with the Y788A substitution but near wild type levels of binding with the more conservative Y788F variant. As predicted from these observations, we have determined that a cytoplasmic splice variant lacking the NP XY sequence is incapable of supporting constitutive PAC1 binding. Splice variants of lacking its NP XY motifs would also be predicted to be unresponsive to inside-out signaling mechanisms.


Figure 6: Alignment of cytoplasmic sequences. Listed above are the wild type cytoplasmic sequences of the , , , , , and subunits. Those residues whose substitution abolished constitutive PAC1 binding are indicated by an asterisk, while those residues whose substitution results in a reduced ability to bind PAC1 are boxed.



A second internalization motif, originally identified in the transferrin receptor, is Y XRF (42) . Molecular modeling (42) and NMR analysis of wild type peptides (43) suggest that both NP XY and YTRF form a reverse turn conformation. This conformation is hypothesized to present a recognition domain to adaptins in clathrin-coated pits thereby mediating uptake (43) . Interestingly, peptides derived from receptors defective in endocytosis do not form the turn conformation. In our studies, the substitution of NPIY for YTRF in the tail completely abolished PAC1 binding. Since this substitution might not disrupt overall structure, the abolition of PAC1 binding is probably related to the loss of specific NPIY residues. It is conceivable that the NP XY motif could represent a recognition site for regulatory, intracellular moieties. Interestingly peptides spanning this region also inhibit talin-integrin interactions in vitro (44) . Thus, our results suggest a novel role for the membrane-proximal NP XY motif in the regulation of ligand binding. Meanwhile, the role of the NP XY motif in integrin internalization is ambiguous. Expression of a truncation that lacks its two NP XF motifs or an F-A substitution variant in the membrane-proximal NP XF both result in a loss of internalization (45) . In contrast, substitution of the tyrosines for serines in the two NP XY motifs of had no effect on internalization (46) . Finally, the NP XY motifs of do not mediate internalization of .()

In contrast, amino acid substitutions within the distal NP XY motif of (N797I, Y800A, Y800F) had a lesser effect on receptor affinity. Similarly, a mutation in (Y759A) analogous to Y800A shows a reduced but variable ability to bind PAC1. Thus these residues do not seem to be nearly as crucial for the maintenance of high affinity binding as those in the first NP XY. Consistent with these results, this motif is not nearly as well conserved amongst cytoplasmic sequences. As noted above, only and possess an exact distal NP XY/F sequence. While these sequences may play a minor role in the regulation of binding affinity, this motif in is required for cellular adhesion. Substitution of phenylalanine in the distal NP XF of this subunit abolished -intracellular adhesion molecule 1 (ICAM-1) interactions (5) .

The phosphorylation of integrin tails has been proposed to play a role in affinity modulation. In our studies, two variants which completely block PAC1 binding, S752P and Y788A, also represent potential phosphorylation sites. However, more conservative, yet phosphorylation-defective, substitutions at these residues (S752A and Y788F) results in greater or near wild type levels of PAC1 binding (Figs. 3 and 4). Thus the abolition of binding by S752P and Y788A is due to an effect other than on phosphorylation. Similarly, other (S790M, S790D, T793V/T794V, Y800A) and (Y747A, Y759A) variants, which eliminate potential phosphorylation sites, retain measurable levels of PAC1 binding. These results argue strongly against a role of phosphorylation in the enhancement of binding affinity and are consistent with others that discount a role for phosphorylation in promoting cellular adhesion (5, 14, 47, 48) .

It is apparent that mutations at several sites within the tail reduced or abolished PAC1 binding. In addition to those variants discussed above, (F771L, E774V) and (F727A/F730L/E733V) substitutions within the putative -actinin binding site (7) also reduced constitutive PAC1 binding. One explanation for these observations is that the dynamic regulation of ligand binding does involve many sites in the tail. Alternatively, tail folding and conformation may be extremely sensitive to amino acid substitution and it is these conformational changes which disrupt normal signaling pathways.

It is also noteworthy that similar qualitative effects on PAC1 binding were observed whether the or point mutations were coexpressed with chimeras containing the , or tails. It is possible that high affinity binding specified by these different tails might involve a single regulatory pathway and thus the same cytoplasmic sequence elements. Alternatively, each integrin may be independently regulated yet utilize the same sequence elements. In this latter case integrin specific regulation would be mediated by sequences in the chain. Distinguishing between these possibilities and identifying the mechanisms of cell-specific affinity modulation awaits further study.

In summary, we have defined a subset of cytoplasmic residues required for inside-out signaling. Substitutions within a membrane-proximal NP XY motif were the most effective in the abolition of binding, suggesting a novel function for this sequence. Defining the interaction of intracellular elements with these sequences will contribute to an understanding of the mechanisms of affinity modulation.


FOOTNOTES

*
This work was supported in part by University of California Tobacco Related Disease Research Program Grants 3RT-0320 and HL-48728. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 619-554-7151; Fax: 619-554-6403; E-mail: otoole@scripps.edu.

The abbreviations used are: CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate; LDL, low density lipoprotein.

J. Ylanne, J. Huuskonen, T. E. O'Toole, M. H. Ginsberg, I. Virtanen, and C. G. Gahmberg, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Mark Ginsberg for suggestions and critical review of the manuscript.


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