Prediction of protein structural class by discriminant analysis

Protein structural class--alpha, beta, mixed (alpha/beta or alpha + beta), irregular--can be predicted from the amino acid sequence by discriminant analysis. Discrimination is based on distributions, in the classes, of vectors of attributes characterizing the sequences. In this paper, two sets of attributes and two methods of estimating their distributions are compared using more than 100 proteins from the Protein Data Bank. The best results were obtained when canonical variates of the frequencies of occurrence of 20 amino acids and non-parametric estimates of their distributions were used. Three variates are sufficient to allocate proteins to one of four classes with 83% reliability (estimated by cross-validation) and four variates allowed allocation to one of five classes with 78% reliability.     

A comparison of two ATPase based schemes for histochemical muscle fibre typing in various mammals

A comparative study was performed on the fibre populations in tibialis anterior muscles of mouse, rat, guinea pig, rabbit, cat and dog using the two different methods of histochemical staining for myofibrillar ATPase after acid (Brooke and Kaiser 1970) or alkaline preincubations (Guth and Samaha 1970). For all species a complete correspondence existed between type I (Brooke and Kaiser 1970) and beta fibres (Samaha et al. 1970). Gross correspondence (greater than 85%) existed between IIA and IIB (Brooke and Kaiser 1970) and alpha beta and alpha fibres (Samaha et al. 1970) respectively in mouse, guinea pig, rabbit, cat and dog. In the case of mouse and dog, this high degree of correspondence was based on the assumption that mouse tibialis anterior contains no type I and the dog no type IIB fibres. For the rat, a pronounced overlap existed between IIA fibres on the one hand and alpha beta and alpha fibres on the other hand as well as between IIB fibres and alpha beta and alpha fibres. These observations lead to the conclusion that the two classification schemes are not interchangeable for all species and that the two terminologies should be used only in relation with the methods from which they were derived.     

FUZZY SET THEORY APPLIED TO PRODUCT CLASSIFICATION BY A SENSORY PANEL1

It is frequently impossible to meet the assumptions underlying the statistical approach to classification of food products by a sensory panel. To find an alternative, we have investigated the applicability of the fuzzy set theory. Within a fuzzy set framework it is acceptable that a product belongs to several classes simultaneously and no assumptions regarding the distribution of sensory properties for a product class are made. Fuzzy classification models can be constructed from a set of training objects by linking the soft class labels to the sensory attributes applying an inference procedure based on fuzzy logic. A number of fuzzy inference procedures has been evaluated using a number of attribute sets. A satisfactory classification has been found using a very simple implication rule and a set of three attributes.     

Use of conditional probabilities for determining relationships between amino acid sequence and protein secondary structure.

The conditional probability, P(sigma/x), is a statement of the probability that the value of sigma will be found given the prior information that a value of x has been observed. Here sigma represents any one of the secondary structure types, alpha, beta, tau, and rho for helix, sheet, turn, and random, respectively, and x represents a sequence attribute, including, but not limited to: (1) hydropathy; (2) hydrophobic moments assuming helix and sheet; (3) Richardson and Richardson helical N-cap and C-cap values; (4) Chou-Fasman conformational parameters for helix, P alpha, for sheet, P beta, and for turn, P tau; and (5) Garnier, Osguthorpe, and Robson (GOR) information values for helix, I alpha, for sheet, I beta, for turn, I tau, and for random structure, I rho. Plots of P(sigma/x) vs. x are demonstrated to provide information about the correlation between structure and attribute, sigma and x. The separations between different P(sigma/x) vs. x curves indicate the capacity of a given attribute to discriminate between different secondary structural types and permit comparison of different attributes. P(alpha/x), P(beta/x), P(tau/x) and P(rho/x) vs. x plots show that the most useful attributes for discriminating helix are, in order: hydrophobic moment assuming helix greater than P alpha much greater than N-cap greater than C-cap approximately I alpha approximately I tau. The information value for turns, I tau, was found to discriminate helix better than turns. Discrimination for sheet was found to be in the following order: I beta much greater than P beta approximately hydropathy greater than I rho approximately hydrophobic moment assuming sheet. Three attributes, at their low values, were found to give significant discrimination for the absence of helix: I alpha approximately P alpha approximately hydrophobic moment assuming helix. Also, three other attributes were found to indicate the absence of sheet: P beta much greater than I rho approximately hydropathy. Indications of the absence of sigma could be as useful for some applications as the indication of the presence of sigma.     

Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.     

Functional classification of ADAMs based on a conserved motif for binding to integrin alpha 9beta 1: implications for sperm-egg binding and other cell interactions

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are alpha(9)beta(1) and alpha(v)beta(3), and they bind in an RGD-independent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin alpha(9)beta(1) and alpha(v)beta(3) recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg(481) and the Asp-Leu-Pro-Glu-Phe residues (residues 488-492) were critical for alpha(9)beta(1) binding, but the RGD motif (residues 484-486) was not. In contrast, the RGD motif was critical for alpha(v)beta(3) binding, but the other residues flanking the RGD motif were not. As the RX(6)DLPEF alpha(9)beta(1) recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that alpha(9)beta(1) may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that alpha(9)beta(1) bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin alpha(9)beta(1), applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and alpha(9)beta(1) have similar binding specificities for ADAMs and that alpha(9)beta(1), or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As alpha(9)beta(1) is a receptor for many ADAM disintegrins and alpha(9)beta(1) and ADAMs are widely expressed, alpha(9)beta(1)-ADAM interaction may be of a broad biological importance.     

Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form.     

Expression of laminin chains by central neurons: analysis with gene and protein trapping techniques

Laminins exert numerous effects on neurons in vitro, but expression of laminin subunit genes by neurons in vivo remains controversial. To reexamine this issue, we generated mice from ES cells in which the laminin alpha1, alpha5, beta1, and gamma1 genes had been "trapped" by insertion of a histochemically detectable selectable marker, betageo (beta-galactosidase fused to neomycin phosphotransferase). The presence of laminin-betageo fusion proteins was assayed histochemically and immunochemically, revealing expression of laminin beta1 and gamma1 genes, but not alpha chain genes, by defined subsets of neurons in brain and retina. We also used the gene traps in a novel way to assay expression of endogenous laminin subunits, which were barely detectable by ordinary immunohistochemical methods. The trapping vector included a transmembrane domain that anchors proteins otherwise destined for secretion. Laminin alpha/beta/gamma heterotrimers are assembled intracellularly, and we show that the trapped laminin gamma1 fusion protein "co-trapped" endogenous beta1 intracellularly. The laminin gamma1 fusion was also able to co-trap transgene-derived alpha chains, but we detected no co-trapped endogenous alpha chains. The co-trapping method may be generally useful for identifying proteins or isolating protein complexes associated with trapped gene products.     

Random coil structures in bacterial proteins. Relationships of their amino acid compositions to flanking structures and corresponding genic base compositions

In this study we classified regions of random coil into four types: coil between alpha helix and beta strand, coil between beta strand and alpha helix, coil between two alpha helices and coil between two beta strands. This classification may be considered as natural. We used 610 3D structures of proteins collected from the Protein Data Bank from bacteria with low, average and high genomic GC-content. Relatively short regions of coil are not random: certain amino acid residues are more or less frequent in each of the types of coil. Namely, hydrophobic amino acids with branched side chains (Ile, Val and Leu) are rare in coil between two beta strands, unlike some acrophilic amino acids (Asp, Asn and Gly). In contrast, coil between two alpha helices is enriched by Leu. Regions of coil between alpha helix and beta strand are enriched by positively charged amino acids (Arg and Lys), while the usage of residues with side chains possessing hydroxyl group (Ser and Thr) is low in them, in contrast to the regions of coil between beta strand and alpha helix. Regions of coil between beta strand and alpha helix are significantly enriched by Cys residues. The response to the symmetric mutational pressure (AT-pressure or GC-pressure) is also quite different for four types of coil. The most conserved regions of coil are “connecting bridges” between beta strand and alpha helix, since their amino acid content shows less strong dependence on GC-content of genes than amino acid contents of other three types of coil. Possible causes and consequences of the described differences in amino acid content distribution between different types of random coil have been discussed.     

Prediction of distant residue contacts with the use of evolutionary information

In this work we present a novel correlated mutations analysis (CMA) method that is significantly more accurate than previously reported CMA methods. Calculation of correlation coefficients is based on physicochemical properties of residues (predictors) and not on substitution matrices. This results in reliable prediction of pairs of residues that are distant in protein sequence but proximal in its three dimensional tertiary structure. Multiple sequence alignments (MSA) containing a sequence of known structure for 127 families from PFAM database have been selected so that all major protein architectures described in CATH classification database are represented. Protein sequences in the selected families were filtered so that only those evolutionarily close to the target protein remain in the MSA. The average accuracy obtained for the alpha beta class of proteins was 26.8% of predicted proximal pairs with average improvement over random accuracy (IOR) of 6.41. Average accuracy is 20.6% for the mainly beta class and 14.4% for the mainly alpha class. The optimum correlation coefficient cutoff (cc cutoff) was found to be around 0.65. The first predictor, which correlates to hydrophobicity, provides the most reliable results. The other two predictors give good predictions which can be used in conjunction to those of the first one. When stricter cc cutoff is chosen, the average accuracy increases significantly (38.76% for alpha beta class), but the trade off is a smaller number of predictions. The use of solvent accessible area estimations for filtering false positives out of the predictions is promising.     

A model-based approach to Bayesian classification with applications to predicting pregnancy outcomes from longitudinal beta-hCG profiles

This paper discusses Bayesian statistical methods for the classification of observations into two or more groups based on hierarchical models for nonlinear longitudinal profiles. Parameter estimation for a discriminant model that classifies individuals into distinct predefined groups or populations uses appropriate posterior simulation schemes. The methods are illustrated with data from a study involving 173 pregnant women. The main objective in this study is to predict normal versus abnormal pregnancy outcomes from beta human chorionic gonadotropin data available at early stages of pregnancy.     

Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins).

Here we identified several new integrin/TM4 protein complexes on the cell surface. By immunoprecipitation using nonstringent conditions, and by reciprocal immunoprecipitation, we found that alpha 3 beta 1 and alpha 6 beta 1 integrins but not alpha 2 beta 1, alpha 5 beta 1, or alpha 6 beta 4 integrins associated with CD9 and CD81 in alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, alpha 6 beta 1/CD81, and alpha 6 beta 1/CD9 complexes. Also, cross-linking experiments established that alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, and alpha 3 beta 1/CD63 associations occur on the surface of intact cells and suggested that a critical interaction site is located within extracellular domains. Cross-linking in conjunction with reimmunoprecipitation indicated that larger multi-component alpha 3 beta 1/TM4/TM4 complexes (alpha 3 beta 1/CD9/CD63, alpha 3 beta 1/CD81/CD63, and alpha 3 beta 1/CD9/CD81) also could be detected on the cell surface. Immunofluorescent staining showed redistribution of alpha 3 beta 1/TM4 complexes toward the periphery of cells plated on various extracellular matrix substrates and also showed that these complexes were localized in cell footprints. Staining of human tissues yielded additional results consistent with co-localization of alpha 3 beta 1 and CD9, CD63, and CD81 proteins. In conclusion we suggest that the prevalence of integrin/TM4 complexes in diverse cellular environments is indicative of their general physiological importance.     

A quadratic discriminant analysis of protein structure classification based on the Helix/Strand content

Based on the 210 non-homologous proteins (domains) classified manually by Michie et al. (J. Mol. Biol. 262, 168-185, 1996), a new structure classification criterion of globular proteins relying on the content of helix/strand has been proposed, using a quadratic discriminant method. Each protein is classified into one of the three classes, i.e. those of alpha class, beta class and alphabeta class (including alpha/beta and alpha+beta classes). According to the new structure classification criterion, of the 210 proteins in the training set, 207 are correctly classified and thus the accuracy is 207/210=98.57%. Multiple cross-validation tests are performed. The jackknife test shows that of the 210 proteins 207 are correctly classified with an accuracy of 98.57%. To test the method further, of 3577 proteins (domains) extracted from SCOP, 91.39% of them are correctly reclassified by the new classification criterion. On average, the accuracy of the new criterion is about 8 percentage points higher than that of the criterion proposed by Nakashima et al. (J. Biochem. 99, 153-162, 1986). Our result shows that the classification based solely on structures is basically consistent with that combining both structural and evolutionary information. Further complete automated classification scheme should consider both structures and evolutionary relationship. The methodology presented provides an appropriate mathematical format to reach this goal.     

Inhibitory effects of MLDG-containing heterodimeric disintegrins reveal distinct structural requirements for interaction of the integrin alpha 9beta 1 with VCAM-1, tenascin-C, and osteopontin

The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.     

Thermal repair of tryptophan synthase mutations in a regulatory intersubunit salt bridge. Evidence from arrhenius plots, absorption spectra, and primary kinetic isotope effects

This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium. Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge between alpha subunit Asp(56) and beta subunit Lys(167) play allosteric roles. Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperature in combination with CsCl or with NaCl plus an alpha subunit ligand, alpha-glycerol 3-phosphate. Arrhenius plots of the activity data under these conditions were nonlinear. The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. We correlate the results with a model in which the mutant enzymes are converted by increased temperature from a low activity, "open" conformation to a high activity, "closed" conformation under certain conditions. The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms. The results suggest that alpha subunit Asp(56) and beta subunit Lys(167) are not essential for catalysis and for allosteric communication between the alpha and beta subunits but that their mutual interaction is important in stabilization of the active, closed form of the alpha(2)beta(2) complex.     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

The beta subunit of E. coli glycyl-tRNA synthetase plays a major role in tRNA recognition.

The contributions made by the alpha and beta subunits of E. coli glycyl-tRNA synthetase to the recognition of tRNA have been investigated via binding and immunological methods. Using the nitrocellulose filter assay, we have shown that isolated beta subunit, but not the alpha subunit, binds [14C]glycyl-tRNA with an affinity comparable to that of the native enzyme. Further, the data indicate that the beta subunit possesses one binding site for glycyl-tRNA while the native or reconstituted enzyme (alpha 2 beta 2) has two sites. Rabbit antibodies directed at the beta subunit or the holoenzyme inhibit efficiently the ability of the enzyme to aminoacylate tRNA while alpha-subunit antibodies have a smaller effect. Since none of the antisera have an appreciable effect on the ATP-PPi exchange activity of the enzyme under these conditions, the beta-subunit (and holoenzyme) antisera evidently interfere with productive tRNA binding. Taken together, the data indicate that the larger, beta subunit of glycyl-tRNA synthetase plays a major role in tRNA recognition.     

Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.