©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Cytoplasmic Domain of
A TERNARY COMPLEX OF THE INTEGRIN alpha AND beta SUBUNITS AND A DIVALENT CATION (*)

(Received for publication, August 28, 1995; and in revised form, November 20, 1995)

Thomas A. Haas (§) Edward F. Plow

From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Peptides corresponding to the cytoplasmic tails of the alpha (alpha(985-1008)) and beta(3) (beta(3)(713-762)) subunits of the integrin receptor alphabeta(3) (glycoprotein IIb-IIIa) were synthesized and used to characterize their interaction with cations and with one another. alpha(985-1008) was found to contain a functional cation binding site as assessed by both terbium luminescence and electrospray ionization mass spectroscopy. The binding of Tb to alpha(985-1008) was of high affinity (K = 8.8 ± 5.2 nM), occurred with a 1:1 stoichiometry, and was mediated by its acidic carboxyl terminus (alpha(999-1008), PLEEDDEEGE). The affinity of this site for divalent cations was in the micromolar range, suggesting that this site would be constitutively occupied in the intracellular environment. Incubation of alpha(999-1008) with beta(3)(713-762) resulted in the formation of a complex, both in the presence and absence of cations. The interactive site for alpha(999-1008) in beta(3) was mapped to beta(3)(721-740), and complex formation was associated with a stabilization of secondary structure as assessed by circular dichroism. Both a binary (alpha(985-1008)bulletbeta(3)(721-740)) and a ternary (Tbbulletalpha(999-1008)bulletbeta(3)(721-740)) complex were detected by mass spectroscopy, but the distribution and intensity of the mass/charge peaks were distinct. These difference may reflect the involvement of distinct cation coordination sites and the formation of salt bridges in stabilizing the ternary complex. These data demonstrate the formation of a novel entity composed of the cytoplasmic tails of alpha and beta(3) and a cation which may constitute a functional intracellular domain.


INTRODUCTION

The integrin family of receptors mediate many of the cellcell and cell-substratum interactions that are central to cell adhesion, migration, growth, and differentiation. Integrins are noncovalent alpha/beta heterodimers; each subunit contains a large extracellular region of several hundred amino acids, a transmembrane domain, and a single, short (usually less than 50 amino acids) cytoplasmic tail(1, 2, 3, 4, 5, 6, 7) . The extracellular regions of the subunits interact with each other to form a binding site for a wide variety of ligands, including extracellular matrix proteins, counter-receptors on other cells, and circulating plasma proteins(8, 9) . Numerous studies support a model in which amino acid sequences in both subunits coordinate ligand and cations within close proximity to form a ``reactive center'' for ligand binding(10, 11, 12, 13) .

The affinity of integrins for their ligands is tightly regulated through a process termed inside-out signaling(5, 14, 15) . Accordingly, intracellular signals initiate conformational changes, and the extracellular domains can be transformed from a low to a high affinity ligand binding state. The cytoplasmic tails of integrins must be centrally involved in initiating and propagating the conformational changes that mediate such inside-out signaling. At the same time, binding of ligands to the extracellular domain elicits intracellular responses (outside-in signaling), and activation of such signaling pathways must be dependent upon integrin cytoplasmic tails and their conformation(16) . In addition, the cytoplasmic tails serve as binding sites for intracellular ligands, including cytoskeletal proteins such as talin and alpha-actinin(17, 18) . The transmission of conformational change to the cytoplasmic tails must also be centrally involved in such signaling. Thus, the cytoplasmic tails of integrin alpha and beta subunits play a pivotal role in regulating integrin function.

The platelet integrin alphabeta(3) (glycoprotein IIb-IIIa) provides evidence for both inside-out and outside-in signaling and for the importance of its cytoplasmic tails in these processes(19, 20, 21, 22, 23) . On resting platelets alphabeta(3) is in a ``latent'' and/or noncompetent state, as it does not bind its abundant blood-borne ligands, such as fibrinogen(24, 25, 26) . Platelet stimulation with agonists, such as thrombin and ADP, induces inside-out signaling which leads to activation of alphabeta(3) to a competent receptor. Outside-in signaling is manifest by numerous intracellular changes, including the initiation of cytoskeletal reorganization, activation of kinases and phosphatases, and translocation of intracellular constituents(23, 27, 28) . Several lines of evidence directly support the involvement of the cytoplasmic tails of alpha and beta(3) in these events. A single point mutation within the beta(3) cytoplasmic tail, serine 752 to proline, prevents alphabeta(3) from becoming a competent receptor(21) . More recently, it has been reported that transfected cells expressing this beta(3) substitution in alphabeta(3) have impaired alphabeta(3)-mediated cell spreading, focal adhesion, and fibrin clot retraction, suggesting that this mutation also affected outside-in signaling(29) . Truncation of the cytoplasmic tail of alpha results in a constitutively active alphabeta(3)(20) , direct evidence of inside-out signaling.

The central features of integrin extracellular domains and their ligand binding sites extend to alphabeta(3): the subunits interact with one another and bind divalent cations, and the ligand binding function is conformationally regulated. In a sense, the cytoplasmic tails of alphabeta(3) and other integrins can be viewed as a second ligand binding domain by virtue of their interactions with cytoskeletal proteins and intracellular signaling molecules and by their predicted conformational sensitivity. In the present study, we have put this analogy to a direct test. Interaction of the cytoplasmic tails with one another and with cations are demonstrated; and, in turn, these interactions are shown to effect conformation. These findings may have substantial bearing on integrin structure and function.


EXPERIMENTAL PROCEDURES

Materials

HPLC (^1)grade acetonitrile and methanol and peptide synthesis grade N,N-dimethylformamide were obtained from Fisher. Terbium chloride (TbCl(3)bullet6H(2)O), p-cresol, p-thiocresol, and trifluoroethanol (NMR grade) were obtained from Aldrich. tert-Butyloxycarbonyl (Boc)-Asp(OcHx) and Boc-Glu(OcHx) amino acids were obtained from Novabiochem (La Jolla, CA). All other Boc-amino acids, as well as diisopropylethylamine, all peptide synthesis resins, and trifluoroacetic acid (for HPLC) were obtained from Advanced Chemtech (Louisville, KY). Trifluoroacetic acid used in peptide synthesis was obtained from Halocarbon (River Edge, NJ); 1-hydroxybenzotriazole tetramethyluronium hexafluorophosphate (HBTU) was from Richelieu Biotechnologies (Montreal, QC); and ninhydrin reagents were from Applied Biosystems (Foster City, CA).

Peptide Synthesis

All peptides used in this study were synthesized using an automated and a manual stepwise in situ neutralization/HBTU protocol for Boc chemistry solid phase peptide synthesis(30) . The efficiency of coupling at each step was monitored using the quantitative ninhydrin method(30) . Peptides were synthesized either on 4-methylbenzhydrylamine-resins (alpha-carboxyamides) or on appropriate Boc-aminoacyl-OCH(2)-Pam-resins (carboxylates). Dinitrophenol, Boc, and formyl-protecting groups were removed prior to peptide cleavage, and full side chain deprotection was performed in liquid hydrogen fluoride containing 2.5% p-cresol and 2.5% p-thiocresol.

Peptide Purification and Characterization and Solubility

All analytical and semipreparative gradient HPLC were performed on a Gilson 306 dual pump HPLC system equipped with a 811C dynamic mixer and a 117 dual wavelength detector (Gilson Inc., Middleton, WI). Preparative gradient HPLC was performed on a Prep 4000 HPLC system (Waters, Milford, MA) equipped with a 486 tunable wavelength detector (usually set to either 214 or 280 nm). All HPLC runs were performed on Vydac C18 columns. Analytical runs were performed on a 5-µm, 3.8 times 250-mm column at a flow rate of 1 ml/min; semipreparative runs on a 10-µm, 10 times 250-mm column at a flow rate of 3.8 ml/min; and preparative runs on a 15-20-µm, 50 times 250 mm column at a flow rate of 50 ml/min. Linear gradients of water:trifluoroacetic acid (999:1) versus acetonitrile:water:trifluoroacetic acid (900:99.1:0.9) were used in all HPLC runs.

Crude peptides were dissolved in 10% aqueous acetic acid, diluted 1:20 with 5% aqueous acetonitrile (containing 0.1% trifluoroacetic acid), and finally purified by either semipreparative or preparative HPLC. Following lyophilization, the purity of each peptide was confirmed to be >98% as assessed by both analytical HPLC and ESIM spectroscopy. The molecular mass of each peptide, obtained from its ESIM spectrum, was identical to its expected mass, determined using MacProMass (PESciex, Thornhill, ON).

In preliminary studies, the beta(3) peptides tended to precipitate within 2 h when dissolved in buffers containing geq20 mM KCl or NaCl. Therefore, all experiments reported here were performed in buffers containing 10 mM KCl, unless otherwise specified.

Peptide Quantification and Nomenclature

Peptide concentrations were determined by either tryptophan absorbance at 280 nm or 289 nm ( = 5690 M cm and 4850 M cm, respectively), tyrosine absorbance at 276 nm ( = 1450 M cm), or by dry weight.

For peptide nomenclature, the subunit identification (alpha or beta) is followed by the sequence position, with residue 1 being the NH(2) terminus of the mature subunit(31, 32) . Peptides that contained the natural COOH terminus of the subunit was synthesized with a terminal carboxylate; otherwise the carboxyl-terminal residue was in an amide form. The peptides used in this study are as follows: alpha(985-1008), LAMWKVGFFKRNRPPLEEDDE-EGE; alpha(985-998), LAMWKVGFFKRNRP(amide); alpha(999-1008), PLEEDDEEGE; alpha(1008EA), PLEEDDEEGA(amide); alpha(1003DDAA), PLEEAAEEGE(amide); beta(3)(713-762), LIWKLLITIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT; beta(3)(715-737), WKLLITIHDRKEFAKFEEERARA(amide); beta(3)(721-740), IHDRKEFAKFEEERARAKWD(amide); and beta(3)(117-131), LMDLSYSMKDDLWSI(amide).

Terbium Luminescence Spectroscopy

Tb luminescence spectroscopy was performed as described previously(11, 33) . Luminescence measurements were performed on a Jasco FP-777 spectrofluorometer equipped with a thermally regulated cell holder maintained at 20 °C. Unless otherwise stated, all peptides were dissolved in 10 mM KCl, 5 mM PIPES, pH 6.8 (pH adjusted with KOH), containing various concentrations of Tb (Aldrich), and placed in either a 1-cm or a 1-mm rectangular quartz cuvette. A wavelength of 289 nm was used for excitation of tryptophan, and the Tb emission was recorded at 545 nm (no qualitative or quantitative differences were noted when data were collected as either peak area (538-555 nm) or peak height (545 nm)). Both the emission and excitation bandwidths were 3 nm. Emission spectra were corrected manually for blank contribution and automatically for variations in lamp intensity. Preliminary studies showed that cation binding to the reactive peptides was complete within 30 s; therefore, all peptides were incubated for at least 5 min before Tb luminescence was measured.

Tb binding constant for alpha(985-1008) was determined by nonlinear least squares fit of fluorescence intensities to (34) .

In this study, the peptides were dissolved in 5 mM KCl, 5 mM PIPES, pH 6.8. The fluorescence intensities at zero receptor concentration (F(0)) and at receptor saturation (F(s)) and the dissociation constant (K(d)) were all optimized to achieve the best fit of the data as described previously(34) . In addition, a K(d) was also calculated using experimental values for F(0) and F(s). The Tb concentration, [L](0), was kept constant at 300 nM, while the concentration of alpha(985-1008), [R](0), was varied from 50 nM to 10 µM.

The binding of other cations to the peptides under analysis was assessed by their capacity to displace Tb complexed with a peptide, measured as a decrease in the luminescent signal at 545 nm (33) . The K(d) values for these other cations were derived from their Tb displacement curves using the calculated K(d) for Tb (see above). For these calculations, the K(d) was the concentration that decreased the fluorescence intensity by 50%, assuming that [cation] = [cation] - 0.5[R].

Distance measurements using fluorescence energy transfer from tryptophan residues within the peptides to bound Tb were calculated with the following equations(35, 36) : E = 1 - F/F(D) and r = [E - 1]R(0), where E is the efficiency of energy transfer from donor to acceptor; F and F(D) are the fluorescence intensity of the donor in the presence and absence of the acceptor, respectively; R(0) is the Förster critical distance at which E = 0.5 and r is the distance between donor and acceptor. F and F(D) were measured at 350 nm, the emission maximum for tryptophan within the Tb-binding peptide, alpha(985-1008).

ESIM Spectroscopy

ESIM spectroscopy was performed as described previously (11) on an APIII triple quadrupole mass spectrometer (PESciex, Thornhill, ON) using standard operating conditions. Standard operating conditions were not altered in an attempt to stabilize peptide-peptide or peptide-cation complexes. Peptides (10 µM) were dissolved in water with or without 20 µM Tb and injected into the mass spectrometer at a flow rate of 3 µl/min using a Hamilton syringe pump. Full-scan spectra (typically m/z 400-1600) were acquired at a m/z 0.2 accuracy, and the spectra shown represent an average of ten scans.

Tryptophan Fluorescence Spectroscopy

Tryptophan fluorescence spectroscopy was performed using the same instrumentation as for Tb luminescence studies. The peptides (10 µM) were dissolved in 5 mM phosphate, pH 7.2, containing ± 1 M guanidine hydrochloride (GuHCl) and incubated on ice for 60 min. The samples were then placed in a 1-cm rectangular quartz cuvette, thermally regulated to 4 °C. The samples were then excited at 289 nm, and the emission spectra were collected from 300 to 450 nm using 0.5-nm steps. Both the emission and excitation bandwidths were 3 nm. Emission spectra were corrected manually for blank contribution and automatically for variations in lamp intensity.

Circular Dichroism Spectroscopy

Circular dichroism (CD) spectra of the peptides were obtained as previously reported(11, 33) . Briefly, CD measurements were performed on a Jasco J600 spectropolarimeter equipped with a thermally regulated cell holder maintained at 4 °C. Peptides were dissolved in 1 mM KCl, 5 mM phosphate, buffered to either pH 7.2 or pH 3.2 with KOH, and incubated for 30 min on ice. Samples were placed in a 2-mm rectangular quartz cuvette, and the region from 260 to 182 nm was scanned every 0.2 nm using a bandwidth of 0.5 nm. All samples were scanned five times, and the average of these scans were analyzed to obtain a high signal-to-noise representation of the spectrum. Each spectrum was corrected for solvent contribution (scanned in an identical manner and using the same buffer). The data were digitized, expressed in units of mean residual molar ellipticity (Q, degreebulletcm^2bulletdmol), exported into Peakfit (Scanalytics), and then smoothed using the FFT transformation. The data were plotted using the Harvard Graphics Windows program.

Secondary Structural Analysis of CD Spectrum

Secondary structure was estimated as a linear combination of four secondary structural component spectra using 1-nm steps from 240 to 195 nm, using the equation: C = S(R)f(k)S(k), in which C is the CD spectrum of the peptide, and f(k) is the fraction of the k secondary structural component spectra, S(k)(37) . A program, developed in-house and written in Window's Visual Basic language, was used to estimate the secondary structural fractions by least squares fit for C. One of two constraints was imposed, either 0 leq f(k) leq 1, or 0 leq S(i)f(k) leq 1. The program was developed to be robust with respect to: which constraint was imposed, the wavelength range analyzed, and by solving the equation to maximize the coefficient of determination (R^2) and/or minimize the residual sum of squares (Res SS). R^2 and Res SS were calculated using the following equations.

and

The program solved the equation using four secondary structural component spectra (alpha helical, beta sheet, beta turn, and random/extended coil). These spectra were based on either proteins, different peptides sets, and a combination of peptide and protein components(37) . Validation of the program was established in two ways. First, the program always produced correct results when combinations of the four secondary structural component spectra were mathematically added and entered into the program. Second, the program gave results almost identical to those obtained by Yang et al.(37) and with a deconvolution CD program provided with the J600 instrument for four protein CD spectra (sperm whale myoglobin, egg white lysozyme, bovine pancreas ribonuclease A, and bovine pancreas alpha-chymotrypsin).


RESULTS

Cation Binding Properties of the Cytoplasmic Tails

The cation binding characteristics of alpha and beta(3) cytoplasmic tails were assessed using two independent experimental approaches: Tb luminescence and ESIM spectroscopy. These approaches have been used previously on the intact receptor and segments of its extracellular domain(11, 33) . Peptides encompassing the entire cytoplasmic tail of each subunit, alpha(985-1008) and beta(3)(731-762), the amino-terminal aspect of the alpha cytoplasmic tail, alpha(985-998), and a control extracellular beta(3) cation binding peptide, beta(3)(117-131), were evaluated for their ability to bind Tb. As all four peptides contained at least one naturally occurring tryptophan residue within its sequence, Tb binding was evaluated by exciting their tryptophans at 289 nm and monitoring Tb emission at 545 nm. Under these conditions, a measurable signal would only be obtained if Tb was bound in sufficiently close proximity to a tryptophan in the peptide for fluorescence energy transfer(38, 39) . Initially, Tb luminescence was measured with the peptides at 25 µM and Tb at 50 µM. As shown in Fig. 1, alpha(985-1008) yielded a major luminescent signal, indicative of Tb binding to the peptide. This signal was much greater than that of the control cation-binding peptide, beta(3)(117-131). In contrast, both beta(3)(731-762) and alpha(985-998) showed no evidence of Tb binding.


Figure 1: Binding of Tb to the cytoplasmic tail of alpha as monitored by terbium luminescence spectroscopy. Peptides (25 µM) encompassing the cytoplasmic tails of alpha (alpha(985-1008)) and beta(3) (beta(3)(713-762)), the amino terminus of alpha (alpha(985-998)), and the extracellular calcium-binding segment of beta(3) (beta(3)(117-131)) were dissolved in 5 mM MOPS, pH 6.8, containing 50 µM Tb. After a 5-min incubation, luminescence of Tb bound to the tryptophan containing peptides was induced by excitation at 289 nm, and Tb emission was quantitated at 545 nm. Each data point is a mean of three separate experiments with an average of five measurements of each point in each experiment.



The stoichiometry of Tb binding to alpha(985-1008) was evaluated by adding increasing amounts of Tb to a fixed concentration of peptide (10 µM). The results of three separate experiments are shown in Fig. 2A. In the absence of Tb, alpha(985-1008) displayed no Tb luminescence at 545 nm. Increasing amounts of Tb, in the range of 0.25:1 to 1:1 Tb/peptide ratio, resulted in a linear increase in Tb luminescence (r = 0.998). Increasing the amount of Tb added above the 1:1 ratio did not further increase the luminescent signal. Assuming that all the Tb luminescence was directly related to the amount of complex formed, the stoichiometry of Tb binding to alpha(985-1008) was 1:1. Using the data points from ratios of leq1:1, the K(d) of Tb binding to alpha(985-1008) was estimated to be below 10 nM. Accurate measurement of K(d) from titrations, such as shown in Fig. 2A, required using peptide concentrations below 50 nM, where the signal-to-noise ratio was unacceptable. Therefore, the K(d) for Tb binding to alpha(985-1008) was determined as described by Kuzmic et al.(34) . In these experiments, the concentration of Tb was fixed at 300 nM and the concentration of alpha(985-1008) varied (50 nM to 10 µM). The experimental data points are shown in Fig. 2B. The curve represents the theoretical fit of the data to (see ``Experimental Procedures''), after optimizing F(0), F(s), and K(d)(34) . The optimized values obtained were: F(0) = 0.399 ± 0.377, F(s) = 8.231 ± 2.167, and K(d) = 8.8 ± 5.2 nM. Using experimentally obtained values for F(0) and F(s) (0.380 ± 0.512 and 8.492 ± 1.15, respectively), the K(d) was calculated to be 14.6 ± 6.3 nM. Thus, the two approaches yielded very similar values for the various parameters. For comparison, the K(d) of five cation binding peptides from the extracellular region of alphabeta(3), including that of beta(3)(118-131), fell within the 1-15 µM range(33) .


Figure 2: Characterization of alpha(985-1008)bulletTb complex. A, stoichiometry of the alpha(985-1008)bulletTb complex. alpha(985-1008) (bullet) or beta(3)(117-131) (circle), each at 25 µM, were incubated with increasing amounts of Tb and terbium luminescence measured as in Fig. 1. B, determination of the dissociation constant of Tb for alpha(985-1008). Increasing amounts of alpha(985-1008) were added to a fixed amount of Tb (300 nM). After 5 min, Tb luminescence was measured. Each data point (bullet) is an average of five measurements. The displayed curve (-) represents the theoretical fit of the data (see under ``Experimental Procedures''). C, displacement of Tb from the alpha-(985-1008)bulletTb complex by cations. alpha(985-1008) (30 µM) was incubated for 5 min with Tb (25 µM) and then various concentrations of divalent cations were added (Mn, bullet; Ba, ; Ca, times; Mg, circle). After an additional 5-min incubation, the amount of Tb remaining complex to alpha(985-1008) was assessed by Tb luminescence spectroscopy. As all three monovalent cations (K, Na, and Li), used only at a final displacement concentration of 50 mM, were marked with the same symbols (), as they all gave similarly low levels of displacement.



To determine the cation specificity of alpha(985-1008), the ability of divalent (Ba, Ca, Mn, and Mg) and monovalent (K, Na, and Li) cations to displace Tb from alpha(985-1008) was measured (Fig. 2C). In these displacement experiments, the amount of Tb was nonsaturating (25 µM Tb with 30 µM alpha(985-1008)), such that the decrease in Tb luminescence was proportional to the amount of Tb displaced by the competing ion. A 2000-fold excess (50 mM) of any of the monovalent cations relative to Tb resulted in <20% decrease in Tb luminescence. In contrast, the divalent cations did displace bound Tb from alpha(985-1008). Mn was 100-fold more effective than either Ca or Mg in competing with Tb. The displacement curves shown in Fig. 2C were analyzed to calculate the dissociation constants for the divalent cations for alpha(985-1008) using the K(d) for Tb calculated above. These values are summarized in Table 1and indicate that the cation preference of alpha(985-1008) is Mn Ba > Ca approx Mg monovalents.



The absence of a luminescent signal with alpha(985-998) suggests that the carboxylate-rich carboxyl-terminal aspects (alpha-(999-1008)) must be required for the cation binding properties of alpha. However, alpha(999-1008) does not contain a natural aromatic residue, precluding the use of Tb luminescence to directly test this hypothesis. Therefore, the cation binding properties of alpha(999-1008) were directly measured by ESIM spectroscopy. The results of the ESIM spectroscopy experiments are summarized in Fig. 3, A and B. alpha(999-1008) (10 µM) was dissolved in water containing ± 20 µM Tb. In the absence of Tb, the expected ESIM spectrum for the peptide was obtained (Fig. 3A). Only two major electrospray responses (m/z peaks) were observed. Both these peaks corresponded to those expected for alpha(999-1008) (m/z = 1161.4 and 581.2 at charge states 1+ and 2+, respectively). Addition of Tb to alpha(999-1008) (Fig. 3B) resulted in almost complete loss of the two m/z peaks in the alpha(999-1008) spectrum with the concurrent appearance of a new ESIM spectrum. This spectrum corresponded to that predicted for the alpha(999-1008)bulletTb complex, with the loss of three protons for each Tb bound (m/z = 659.2 and 439.8). Similar results were obtained with alpha(985-1008), the peptide encompassing the entire cytoplasmic tail of alpha (data not shown). In contrast, beta(3)(721-740), IHDRKEFAKFEEERARAKWD, which contained three sequential glutamic acid residues showed no Tb binding by ESIM spectroscopy (Fig. 3C versus three-dimensional). Similarly, alpha(985-998), beta(3)(715-737), and beta(3)(713-762) failed to bind Tb, as did a number of control peptides (RGDW, YQAAIDYIN, and YGSTGVFSSWVDRIEEA), demonstrating the specificity of ESIM spectroscopic methodology in determining the capacity of peptides to bind cations. Thus, we conclude that the cation binding activity of the cytoplasmic tail of alpha resides in its carboxyl-terminal aspects.


Figure 3: Electrospray ionization mass spectroscopic analysis of terbium binding to the alpha and beta(3) cytoplasmic tails. Peptides (10 µM) were dissolved in water containing ±20 µM Tb and infused into the mass spectrometer at a flow rate of 3 µl/min using a Hamilton syringe pump. Spectrum for alpha(999-1008) (A, B) and beta(3)(721-740) (C, D), in the absence (A, C) and presence (B, D) of Tb, are displayed. The predicted electrospray response peaks corresponding to uncomplexed(-) and Tb complexed (+) peptides are identified. beta(3)(715-737) yields similar results similar to those obtained for beta(3)(721-740) (data not shown). Full-scan spectra (400-1600 M/Z) were acquired at a 0.2 m/z accuracy, and the spectra shown represent an average of six to ten scans.



Having established that the cation binding properties of alpha(985-1008) resided within alpha(999-1008) (PLEEDDEEGE, containing a free carboxyl terminus), two mutant peptides were synthesized in an attempt to determine which oxygenated residues were involved in cation coordination. The carboxyl terminus of alpha(999-1008) was mutated to PLEEDDEEGA-amide (alpha(1008EA)), and alpha(1003-1004) was mutated from DD to AA (alpha(1003DDAA), PLEEAAEEGE-amide). Terbium luminescence was used to evaluate the cation binding properties of these mutant peptides. Specifically, since these peptides lacked a tryptophan, we evaluated their cation binding capacity as competitors of Tb binding to alpha(985-1008). Incubation of an equimolar amount of alpha(985-1008) (20 µM) with either alpha(999-1008), alpha(1008EA), or alpha- (1003DDAA), in the presence of 17.5 µM Tb, resulted in a 55 ± 4%, 55 ± 5%, and 1.3 ± 12.5% decrease in Tb luminescence, respectively. Thus, alpha(1008EA) competed as effectively as the control native peptide for Tb binding, but alpha(1003DDAA) bound Tb poorly. These data suggest that neither the carboxylate terminus of alpha nor the carboxylate side chain of glutamate alpha(1008) is required for cation binding, while at least one of the carboxylate side chains of the aspartates at alpha(1003) and/or alpha(1004) is essential for cation binding.

Finally, we attempted to estimate the distance from the tryptophan at alpha(986) to the bound Tb ion. The efficiency of energy transfer from donor to acceptor, E, was determined to be 8.3 ± 0.4% (n = 9). Assuming a Förster critical distance (R(0)) range of 10-22.7 Å for donor-Tb(35, 36, 40) , the range in distance between donor (alpha(988W)) and acceptor (Tb) was calculated to be 15-34 Å.

Interaction between the Cytoplasmic Tails of alphaand beta(3)

Initial evidence for an interaction of the cytoplasmic tails was derived from fluorescence experiments. This approach was similar to that used by Muir et al.(41) to suggest intramolecular interactions within a linked peptide containing alpha and beta(3) cytoplasmic tail sequences joined to helical mimetics of their transmembrane domains. The alpha(985-1008) and beta(3)(713-762) peptides both contain tryptophan residues (1 in the alpha and 2 in beta(3) peptide). The two peptides were mixed together at a equimolar ratio (10 µM) in the presence or absence of 1 M GuHCl to prevent interaction. The tryptophan residues in the peptides were excited at 289 nm, and the emission spectrum was recorded in the 300-450-nm range. Co-incubation of alpha-(985-1008) and beta(3)(713-762) in the absence of GuHCl led to a small, but significant (p < 0.001), quenching of tryptophan emission compared with in the presence of the denaturant (Fig. 4). The spectrum obtained in the presence of GuHCl was identical to that obtained by the mathematically addition of the spectra of the two individual peptides. Moreover, for the individual peptides the fluorescence maximum was linearly related to their concentration in the range of 0.1-100 µM, suggesting that the observed fluorescence quenching was not due to peptide self-association. Thus, the emission signal from at least 1 of the 3 tryptophan residues emission spectrum was quenched, most likely arising from an intermolecular interaction.


Figure 4: Evidence for interaction of the cytoplasmic tails of alpha and beta(3) by fluorescence quenching. alpha(985-1008) and beta(3)(713-762) peptides (10 µM) were dissolved together in 5 mM phosphate, pH 7.2, in the absence or presence of 1 M GuHCl. The peptides were co-incubated for 60 min on ice and then placed in a 4 °C thermally regulated quartz cuvette. The samples were then excited at 289 nm (bandwidth = 3 nm) and the emission spectra recorded from 300 to 450 nm using 0.5-nm steps and a bandwidth of 3 nm. Background contribution was then subtracted from each spectra. The average spectra from five scans of the peptides obtained in the absence (-) and presence (- - - -) of GuHCl are displayed. Similar results to those obtained in the presence of GuHCl were obtained by mathematical addition of the individual spectra of alpha(985-1008) and beta(3)(713-762) (data not shown).



Terbium luminescence provided a more incisive approach to demonstrate interaction between the alpha and beta(3) cytoplasmic tails. In these analyses, we exploited the fact that alpha(999-1008) contains the high affinity cation-binding site (see above), but lacks an aromatic residue for the excitation of bound Tb. However, if the alpha(999-1008)bulletTb binary complex formed a stable ternary complex with a tryptophan-containing peptide, then fluorescence energy transfer could occur, provided that the tryptophan and Tb were in close proximity. Thus, if the tryptophan-containing peptide itself was incapable of binding Tb, then all Tb luminescence would depend upon the formation of the ternary complex. This was found to be the case. When alpha(985-998) or beta(3)(721-740) peptides were incubated with a 1.5 molar excess of Tb, no Tb luminescence signal was detected (Fig. 5A), confirming our ESIM data (Fig. 3) that these peptides do not bind Tb. As expected, alpha(999-1008) did not generate a luminescent signal as it lacks a tryptophan donor (Fig. 5A). However, when alpha(999-1008) was co-incubated with beta(3)(721-740) and Tb, a large luminescence signal was generated (Fig. 5A). These data provide direct evidence for the formation of an alpha(999)bulletbeta(3)(721-740)bulletTb ternary complex. Similar results were also obtained when beta(3)(721-740) was replaced with either beta(3)(713-762) or beta(3)(715-737) (data not shown), suggesting that beta(3)(721-737) encompasses a majority of the alphabulletTb binding domain.


Figure 5: Evidence for interaction of the cytoplasmic tails of alpha and beta(3) by terbium luminescence. A, mapping of the interactive sites in cytoplasmic tails of alpha and beta(3). Individual peptides (25 µM) corresponding to either alpha(985-1008), alpha(985-998), alpha(999-1008), and beta(3)(721-740), or alpha(999-1008) co-incubated with alpha(985-998) (alpha/alpha) and beta(3)(721-740) (alpha/beta), were incubated with 37.5 µM Tb for 30 min before luminescence was measured. B, stoichiometry of alphabulletbeta(3)bulletTb complex as measured by Tb luminescence. alpha(999-1008) (10 µM) was incubated with a 1.5 molar excess of Tb and incubated with increasing amounts of either the beta(3)(721-740) peptide (bullet) or with a control alpha peptide, alpha(985-998) (circle). The resulting terbium luminescence was measured as outlined in the legend to Fig. 1.



The stoichiometry of this ternary complex with respect to its peptide constituents was determined to be 1:1 (Fig. 5B). Varying amounts of beta(3)(721-740) were added to a constant concentration of alpha(999-1008) (10 µM) in the presence of a molar excess of Tb. As shown in Fig. 5B, the maximum Tb luminescence was observed at a 1:1 molar ratio of the two peptides and did not increase as excess beta(3)(721-740) was added. In addition, the interaction of alpha(999-1008) with beta(3)(721-740) was not merely a nonspecific charge-charge interaction between the two peptides, as alpha(999-1008) failed to complex with alpha(985-998) (Fig. 5, A and B), a peptide containing four positively charged residues (three side chains and one free amino terminus).

Conformation Changes Occur during Formation of the Cytoplasmic Tail Complex

Having established that the cytoplasmic tails of alpha and beta(3) interact, we then determined if complex formation resulted in changes in secondary structure. The CD spectra obtained from the equimolar mixture of alpha(985-1008) and beta(3)(713-762) peptides (pH 7.2) is shown in Fig. 6A. This spectrum differed substantially from the mathematically addition of the spectra of the individual peptides (p < 0.001). Most notably, the actual spectrum differed from the theoretical spectrum with respect to both a shift in the location (203-206 nm), and the intensity (-19.0 to -12.9 times 10 degree cm^2 dmol) of its minimum, indicative of an increase in helical content and corresponding decrease in random/extended content. These changes were the same in the presence or absence of Tb (60 µM). Lowering the pH to 3.2 (Fig. 6B) abolished the differences between the actual and theoretical curves, verifying the accuracy of the method for mathematical addition of the curves. Secondary structural predictions indicated that the differences in the mathematical versus actual CD spectra at pH 7.2 could be accounted for by a dramatic decrease in random/extended content (56.1 ± 0.2% versus 28.6 ± 0.3%, respectively) arising from a small increase in alpha helical content (27.2 ± 0.1% versus 32.7 ± 0.1%) and a large increase in beta turn content (16.8 ± 0.2% versus 38.8 ± 0.3%) of the alpha(985-1008)bulletbeta(3)(713-762) complex. Less than 0.1% beta sheet content was found in either spectra.


Figure 6: Stabilization of secondary structure by formation of the alphabulletbeta(3) cytoplasmic tail complex as assessed by circular dichroism. alpha(985-1008) and beta(3)(713-762) peptides (30 µM) were dissolved either individually or together in 5 mM phosphate, buffered to either pH 7.2 (A) or pH 3.2 (B) with 1 M KOH. The peptides were incubated for 30 min on ice and then placed in a 4 °C thermally regulated quartz cuvette, and their CD spectra obtained (an average of five scans). Background contribution was then subtracted from each spectrum, and the spectra obtained for the co-incubated peptides are displayed (-). The spectra obtained from the individual alpha and beta peptides were mathematically added together, and this calculated combined spectrum is displayed(- - - -).



Despite the fact that ESIM measurements are performed in the gaseous phase, ESIM spectrometry has been proven to be a powerful technique for obtaining information about three dimensional protein structure and ligand binding and to be in good agreement with solution phase techniques(11, 42, 43, 44, 45, 46, 47, 48) . This was also the case in our study, the interaction of the cytoplasmic tails with cation and each other as detected by ESIM spectroscopy, were in agreement with Tb luminescence, CD spectroscopy, and fluorescence quenching. Therefore, further information pertaining to the conformation of the binary peptide-peptide and the ternary cation-peptide-peptide complexes were obtained by ESIM spectrometry. When 10 µM alpha(999-1008) and beta(3)(721-740) peptides were co-incubated, a series of electrospray response peaks were generated (Fig. 7A). This series corresponded to the expected m/z peaks for the alpha(999-1008)bulletbeta(3)(721-740) complex (-). In agreement with our terbium luminescence data, both alpha(999-1008)bulletbeta(3)(713-762) and alpha(999-1008)bulletbeta(3)(715-737) complexes could also be generated. The relative abundance of molecular charged ion components of the complex can be determined by comparing the height of each m/z peak. The ``envelope'' of the m/z peaks of the alpha(999-1008)bulletbeta(3)(721-740) complex had one maximum (m/z 941.4, charge state of 4+), and all charge states from 3+ to 8+ were represented. These data demonstrate that none of the charge states were suppressed, which, in turn, indicates that none of the positively charged groups in the complex were masked by the molecular associations. The addition of 20 µM Tb to the complex resulted in the formation of an additional series of peaks (+) corresponding to the Tbbulletalpha(999-1008)bulletbeta(3)(721-740) ternary complex (Fig. 7B). In comparing the intensities of the two series (- versus +), it was evident that the relative abundance of the binary complex(-) was at least 2-fold greater than that of the ternary complex (+), if one assumes that each complex yields a electrospray series of equal intensity. The envelope of the binary complex(-) in the absence versus the presence of Tb (Fig. 7, A versus B, and Table 2) exhibited changes in the overall intensity of m/z peaks, but the shape and the location of the peaks were not altered. Thus, the presence of Tb did not distort the pattern of the binary alphabulletbeta(3) cytoplasmic tail complex. However, when the envelope of the ternary complex (+) (Fig. 7B and Table 2) was examined, the pattern was markedly different. Two maxima were present at m/z 980.4 and 490.7 were observed, corresponding to the 4+ and 8+ charge states, respectively. Furthermore, all charge states were not present. Missing were the expected peaks at m/z 1306.9 (3+) and m/z 560.7 (7+) (the 6+ charge state of the ternary complex (m/z 654) was masked by the 2+ charge state of alpha(999)bulletTb (m/z 659.2)). These distortions indicate alterations in the charges and/or their distribution in the ternary as compared with the binary complexes. Finally, the positions of the m/z peaks shown in Fig. 7, A and B, are consistent with binary and ternary complexes at stoichiometries of 1:1 and 1:1:1. Evidence for higher order complexes was not detected.


Figure 7: Observation of an alphabulletbeta(3) binary and an alphabulletbeta(3)bulletTb ternary complex by electrospray ionization mass spectroscopy. alpha(999-1008) and beta(3)(721-740) peptides (10 µM) were mixed in the absence (A) and presence (B) of 20 µM Tb. After a 5-min incubation, the samples were infused into the mass spectrometer and spectra collected as outlined in the legend to Fig. 3. Spectra corresponding to expected series for the alpha(999-1008)bulletbeta(3)(721-740)(-) and for the alpha(999-1008)bulletbeta(3)(721-740)bulletTb (+) complexes are highlighted. For clarity, all off-scale electrospray response peaks corresponding to either alpha(999-1008), beta(3)(721-740), or alpha(999-1008)bulletTb were deleted from the displayed spectra.






DISCUSSION

In this study, we have examined the interactions of the cytoplasmic tails of alpha and beta(3) with each other and with cations. The data support the following conclusions. First, the carboxyl-terminal aspect of alpha contains a high affinity cation binding site. Second, the two cytoplasmic tails interact with each other to form a binary (peptide-peptide) or a ternary (peptide-peptide-cation) complex. Third, complex formation between the alpha and beta(3) cytoplasmic tails involves their carboxyl- and amino-terminal aspects, respectively. Fourth, as a consequence of cytoplasmic tail interaction, a complex-specific conformation is stabilized. Taken together, these data support a model in which the cytoplasmic portions of the alpha and beta(3) subunits interact with each other and a cation to form the intracellular domain of the receptor.

The negatively charged carboxyl terminus of the alpha cytoplasmic tail was found to be a high affinity cation binding site. This stretch of 10 amino acids, alpha(999-1008), bound Tb with a 1:1 stoichiometry as determined by terbium luminescence and ESIM spectroscopy (Fig. 1Fig. 2Fig. 3). From Tb displacement experiments, we demonstrated that the cation binding properties of this segment was divalent ion specific with a preference for Mn Mg approx Ca. The K(d) for Mn was calculated to be 110 nM and approx15 µM for both Mg and Ca. This cation specificity is different from that of the peptides corresponding to the extracellular cation binding segments of alphabeta(3)(33) , which bind these cations with much lower affinity (K(d) approx mM) and little preference. As the intracellular concentrations of both Mn and Mg(49) are at least 3-100-fold greater than their K(d) for alpha, it is predicted that this site should be constitutively occupied. Constitutive occupancy and/or extremely high affinity of this site for Tb are reasonable explanations for the failure to detect this site in previous analyses of the cation binding properties of intact alphabeta(3)(33, 50) . Using the Förster theory and equations(51) , we calculated that the bound Tb within alpha(985-1008) would be located 15-34 Å from the tryptophan at alpha(988). Recently, we have constructed a molecular model of alpha(985-1008) using the Biosym Insight II software. (^2)Docking of Tb within the carboxyl terminus of the peptide was possible, and the distance to alpha(988W) was 18-26 Å. In our molecular model, the carboxyl terminus of alpha is directed toward the plasma membrane, due to a predicted turn motif at the Pro-Pro sequence (alpha(998-999)). Thus, the Pro-Pro turn motif in alpha brings the bound cation close to the plasma membrane.

Do other integrin alpha subunits contain a cation binding domain? The clustering of charged residues at the carboxyl terminus is unique to alpha. In addition, we have tentatively identified the carboxylate side chains of alpha(1003-1004) as providing at least one key coordination site for cation binding. The alpha(v) cytoplasmic tail also contains a highly charged sequence, QEEQEREQLQPHENGE, but not at its carboxyl terminus. Other integrin alpha subunits appear to lack such sequences, but definitive conclusions cannot be drawn until all the residues that are directly involved in cation coordination in alpha(999-1008) are precisely defined. The cytoplasmic cation binding site in alpha has no homology to the integrin extracellular ``DXSXS'' cation-binding segments(52) , nor to other known cation binding domains(53, 54, 55, 56) . However, the sequence of alpha-(999-1008), EEDDEEGE, is identical to the carboxyl-terminal acidic tail of xUBF Xenopus transcription factor(57) , and it is predicted that this factor also has cation binding properties. Finally, with respect to cytoplasmic tail of beta(3), our combined terbium luminescent and ESIM spectroscopic data indicate that this segment does not bind cation with high affinity in the absence of alpha. Given that the beta subunits are highly homologous, we expect that none of the beta cytoplasmic tails have strong cation binding properties.

Four independent sets of observations, fluorescence quenching, terbium luminescence, ESIM spectroscopy, and CD spectroscopy, indicate that the cytoplasmic tails of alpha and beta(3) interact with each other. In addition to our preliminary report of this observation(58) , two subsequent studies have suggested that the cytoplasmic tails of alpha and beta(3) can interact(41, 59) . In a modeling study, Rocco et al. (59) proposed that the two cytoplasmic tails might interact. Muir et al.(41) designed and synthesized a model protein (MP-1) containing the cytoplasmic tails of both alpha and beta(3) covalently-linked through a constrained helical coiled-coil motif. The coiled-coil motif tertiary structure was incorporated to mimic the transmembrane domains of an integrin receptor, with both cytoplasmic domains aligned in parallel arrangement. In MP-1, the tryptophan emission from beta(3)(739) was protected from heavy metal quenching, which suggested that this residue may be buried in a complex. In our study, using unconstrained free peptides, complex formation was clearly demonstrable. It should be noted that in the linked construct of Muir et al.(41) , the proline-proline (Pro-Pro) amino acid sequence at alpha(998-999) was mutated to histidine-threonine(41) . Although these substitutions may very well alter the conformation of the alpha cytoplasmic tail (in alpha(v), the Pro-Pro sequence is predicted to be a turn motif (60) ), complex formation still occurred, suggesting that a highly constrained conformation for the alpha subunit may not be essential for complexation. Furthermore, as MP-1 contained model transmembrane regions, the combination of data from our study and from that of Muir et al.(41) provide strong evidence that this alpha/beta cytoplasmic interaction occurs in a biological setting, in which alphabeta(3) is inserted into the plasma membrane.

The contact sites in the alpha and beta(3) cytoplasmic tails were mapped to the cation-binding domain of alpha, its carboxyl terminus, and to beta(3)(721-740) ( Fig. 5and Fig. 7). Furthermore, as substitution of beta(3)(721-740) with either beta(3)(713-762) or beta(3)(715-737) yielded similar terbium luminescence and ESIM results, we further concluded that the majority, and most likely all, of sites of contact of alpha(999-1008) for beta(3) must lie between beta(3)(721) and beta(3)(737). This region is highly conserved among the integrin beta subunits. In beta(1), this region encompasses the ``cyto 1'' region, which has been implicated as an alpha-actinin binding site(18, 62) . O'Toole et al.(63) also have implicated this region of beta(3) as regulating integrin inside-out signaling. These observations raise the possibility of a role for cytoskeletal elements in modulating the integrin intercytoplasmic tail interactions, which, in turn, could influence inside-out signaling. At first glance, this possibility appears to be contradicted by the data of Kassner et al.(64) , who demonstrated that a short, nonspecific stretch of amino acids after the highly conserved alpha region, the GFFKR motif, was the only requirement to maintain integrin function in both alpha(4) and alpha(2). However, it should be noted that neither alpha(4) nor alpha(2) contains a potential turn motif in its cytoplasmic tail and are unlikely to bind cation. Furthermore, truncation of alpha cytoplasmic tail before the GFFKR motif resulted in the expression of a constitutively active alphabeta(3) receptor(20) , which was not reversed with either alpha(5) or alpha(6) chimeras, in which the beta(3) recognition sequence in alpha, alpha(999-1008), was destroyed(20, 22) . Thus, the regulation of alpha(4) and alpha(2) integrins may differ from that of alphabeta(3), and cation binding to alpha subunits may individualize integrin functions.

That the cytoplasmic tail complex of alpha and beta(3) forms a conformational entity, distinct from its free constituents, is supported by our fluorescence quenching, CD and ESIM spectroscopic data. In the fluorescence analysis (Fig. 4), we observed quenching, but no shift in the tryptophan emission spectrum from the 350 nm of each individual subunit. The maximum emission for tryptophan in an exposed hydrophilic environment is near 350 nm and is shifted toward the 320-340-nm range in a hydrophobic environment(65) . Thus, it appears that the three tryptophans (alpha(988), beta(3)(715), and beta(3)(739)) remain in a hydrophilic environment and are still partially exposed to solvent in the formed complex. One factor which can quench the intensity of the tryptophan emission spectrum is the proximity of the tryptophan to a carboxylate side chain of either aspartate or glutamate(65, 66) . Accordingly, interaction of the negative carboxyl terminus of alpha with or near one of the tryptophans in beta(3), most likely near beta(3)(739), could account for the fluorescence quenching. From the CD analyses, a small increase in helical content of the alpha and beta(3) cytoplasmic tail complex was evident (Fig. 6). It is most likely that the increase in helical content involves the beta(3) rather than the alpha subunit, based upon our observations that the helical content of beta(3)(713-762), but not that of alpha(985-1008), could be markedly increased by trifluoroethanol, a helix promoting agent; and predictions of the helical content of the beta(3)versus the alpha cytoplasmic tails by molecular modelling.^2 Most interesting was the large increase in beta turn conformation which occurred upon complexation. The alpha and beta cytoplasmic tails are predicted to contain a single turn motif, located at alpha(998-999) (Pro-Pro motif) and beta(3)(744-747) (NPXY motif). The NPXY motif is highly conserved in all beta integrins and has been implicated as an alpha-actinin binding site in beta(1) integrins(18, 62) . Thus, our data raises the possibility that alpha/beta subunit complexation may regulate cytoskeletal attachment and/or vice versa. A functional role for the alpha subunit Pro-Pro turn motif is suggested in our study in establishing subunit interaction. Although this motif is present in only a few alpha subunits, it is present in alpha(V), the other beta(3) alpha subunit.

In addition to the binary peptide-peptide complex, the formation of a cationbulletalphabulletbeta(3) ternary complex was demonstrable by ESIM spectroscopy and terbium luminescence. The stoichiometry of the complexes was 1:1:1 (Fig. 5B and 7B). We did not detect any major differences in the conformation of binary versus the ternary complex by CD. Such was not the case with ESIM spectroscopy, which indicated major differences between the binary and ternary complexes ( Fig. 7and Table 2). The ESIM spectra of the alpha(999)bulletbeta(3)(721-740) binary complex contained all the expected charge states from 2+ to 8+, with a typical ESIM spectrum envelope, indicating that all positive charge groups were exposed. In contrast, the ESIM spectrum envelope of the cationbulletalphabulletbeta(3) ternary complex was clearly not typical, in that it contained two maxima and diminished or missing charge states. These data could arise if positively charged groups were either buried in a hydrophobic core of the complex or were involved in hydrogen bonding and in salt bridges(44, 48) . As the peptides involved are small (leq20 residues) and hydrophilic, the first possibility seems remote. More likely is the possibility that one or more of the charged residues is involved in an interaction, such as forming a salt bridge.

The role of cations in stabilizing protein-protein interactions has become increasingly apparent from structural analyses. The crystal structure of two cation binding domains, the A domain of the alpha subunit of alpha(M)beta(2)(52) and the epidermal growth factor-like domain of human clotting factor IX (54) provide examples in which cations participate directly in and stabilize protein interactions. The distortion of the m/z peaks in the ternary complex could be due to such a rearrangement in cation coordination sites in alpha upon complexation with beta(3). To speculate, complexation of the two cytoplasmic tails would allow one of alpha's cation-coordinating carboxylate side chains to be provided by a residue in beta(3) instead (either by a main chain carbonyl oxygen or by a carboxylate/carboxyamide side chain). The freed carboxylate side chain in alpha could then form the proposed salt bridge with a lysine or arginine side chain in beta(3). This arrangement in cation coordination sites would explain the loss of ESIM peaks and provide a mechanism driving the conformation difference between the ternary and binary complexes. In support of this hypothesis, our molecular models^2 of the alpha and beta(3) cytoplasmic tails do permit interactions of the carboxyl-terminal aspects of alpha with the amino-terminal aspects of beta(3). Such an arrangement is possible because of the predicted beta turns in each cytoplasmic tail. Nevertheless, the same regions of beta(3) also have been implicated in binding multiple intracellular constituents, including cytoskeletal proteins (18) and endonexin(61) . It may be that the different conformational states of the cytoplasmic domain will dictate which interactions are favored. Studies to test this model and to assess the role of complex stabilization and destabilization in initiating and propagating the conformational changes associated with outside-in and inside-out signaling are in progress.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL38292 and HL54924. 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.

§
Supported by a fellowship from the American Heart Association, Northeast Ohio Affiliate.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; Boc, tert-butyloxycarbonyl; ESIM, electrospray ionization mass; HBTU, 1-hydroxybenzotriazole tetramethyluronium hexafluorophosphate; GuHCl, guanidine hydrochloride; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
T. A. Haas and E. F. Plow, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Stephen B. H. Kent (The Scripps Research Institute, La Jolla, CA) for his expertise and guidance in peptide synthesis.


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