Integrin alpha2beta1 is the required receptor for endorepellin angiostatic activity

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the alpha2beta1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the alpha2 I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of alpha2beta1. Microvascular endothelial cells from alpha2beta1(-/-) mice, but not those isolated from either wild-type or alpha1beta1(-/-) mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in alpha2beta1(-/-) mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin alpha2beta1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the alpha2beta1 integrin receptor.     

The amino terminus of G protein alpha subunits is required for interaction with beta gamma

The guanine nucleotide-binding proteins (G proteins), which transduce hormonal and light signals across the plasma membrane, are heterotrimers composed of alpha, beta, and gamma subunits. Activation of G proteins by guanine nucleotides is accompanied by dissociation of the heterotrimer: G + alpha.beta.gamma in equilibrium alpha G + beta.gamma. Brain contains several G proteins of which the most abundant are alpha 39.beta.gamma and alpha 41.beta.gamma. We have used proteolysis by trypsin to study the functional domains of the alpha subunits. In the presence of guanosine 5'-(3-O-thio)triphosphate, trypsin removes a 2-kDa peptide from the amino terminus of these proteins (Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862; Winslow, J. W., Van Amsterdam, J. R., and Neer, E. J. (1986) J. Biol. Chem. 261, 7571-7579). Tryptic cleavage does not affect the GTPase activity of the truncated molecule nor the apparent Km for GTP. However, removal of the 2-kDa amino-terminal peptide prevents association of the alpha subunits with beta.gamma. Since the apparent substrate for pertussis toxin-catalyzed ADP-ribosylation is the alpha.beta.gamma heterotrimer, the trypsin-cleaved alpha subunit is not a substrate for the toxin. Digestion of the carboxyl terminus of alpha 39 with carboxypeptidase A prevents ADP-ribosylation by pertussis toxin but does not interfere with the formation of alpha 39.beta.gamma heterotrimers. We do not yet know whether the amino-terminal region of alpha 39 interacts with beta gamma directly or whether it is necessary to maintain a conformation of alpha 39 which is required for heterotrimer formation. Further studies are needed to define the nature of the contracts between alpha and beta gamma subunits since understanding the structural basis for their reversible interaction is fundamental to understanding their function.     

Complete mapping of divergent amino acids responsible for differential ligand binding of folate receptors alpha and beta

The folate receptor (FR) type alpha may be distinguished from FR-beta by its higher affinity for the circulating folate coenzyme, (6S)-5-methyltetrahydrofolate (5-CH3H4folate), and its opposite stereospecificity for reduced folate coenzymes. Previous studies showed that a single leucine to alanine substitution at position 49 of the mature protein sequence is responsible for the functional divergence of FR-beta (Shen, F., Zheng, X., Wang, H., and Ratnam, M. (1997) Biochemistry 36, 6157-6163); however, the results also indicated that the minimum requirement for conversion of FR-beta to the functional equivalent of FR-alpha should include amino acid substitution(s) downstream of residue 92 in addition to mutation of L49A. To pinpoint those residues, chimeric FR-betaL49A/FR-alpha constructs including progressively shorter segments of FR-alpha downstream of position 92 as well as selected point mutants were studied. Simultaneous substitution of Leu-49, Phe-104, and Gly-166 in FR-beta with the corresponding FR-alpha residues Ala, Val, and Glu, respectively, reconstituted the ligand binding characteristics of FR-alpha. The results also exclude a role for other residues in FR-alpha in determining its functional divergence. A homology model of FR-alpha based on the three-dimensional structure of the chicken riboflavin-binding protein is used to show the position of residues 49, 104, and 166 in relation to the hydrophobic cleft corresponding to the riboflavin-binding pocket.     

From interstellar amino acids to prebiotic catalytic peptides: a review

Amino acids were most likely available on the primitive Earth, produced in the primitive atmosphere or in hydrothermal vents. Import of extraterrestrial amino acids may have represented the major supply, as suggested by micrometeorite collections and simulation experiments in space and in the laboratory. Selective condensation of amino acids in water has been achieved via N-carboxy anydrides. Homochiral peptides with an alternating sequence of hydrophobic and hydrophilic amino acids adopt stereoselective and thermostable beta-pleated sheet structures. Some of the homochiral beta-sheets strongly accelerate the hydrolysis of oligoribonucleotides. The beta-sheet-forming peptides have also been shown to protect their amino acids from racemization. Even if peptides are not able to self-replicate, i.e., to replicate a complete sequence from the mixture of amino acids, the accumulation of chemically active peptides on the primitive Earth appears plausible via thermostable and stereoselective beta-sheets made of alternating sequences.     

Critical amino acid residues of the alpha4 subunit for alpha4beta7 integrin function

A characteristic feature of integrin-ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified in the alpha and beta subunit of the alpha4 integrins, alpha4beta1 and alpha4beta7, and within their ligands which display the tripeptide LDV in fibronectin and homologous motifs in VCAM-1 and MAdCAM-1. The extracellular domain of the murine and human alpha4-subunit contains three conserved LDV motifs, designated LDV-1 to -3. Using site directed mutagenesis and transfection studies, we now examined the functional relevance of the LDV motifs for alpha4beta7 integrins. We present evidence that LDV-1 mutants (D489N) behave like alpha4 wt cells, but LDV-3 mutants (D811N) are impaired in alpha4beta7 integrin-triggered homotypic cell aggregation and in adhesion and spreading on alpha4 specific ligands. Further characterization of LDV-3 mutants revealed a defect in mAb-induced alpha4beta7-cell surface cluster formation. Mutation of the LDV-2 motif (D698N) caused loss of alpha4beta7 integrin cell surface expression. Our results indicate: (i) that LDV-3, located proximal to the cell membrane, is important for alpha4beta7 integrin-triggered functions and for lateral clustering and (ii) that LDV-2 affects alpha4beta7 heterodimer stability.     

From malate dehydrogenase to phenyllactate dehydrogenase. Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity

Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases.     

Key amino acids for differential coupling of alpha1-adrenergic receptor subtypes to Gs

We have established that differing effects of alpha1-adrenergic receptor (AR) subtypes on cell proliferation are due to differential coupling to the Gs/cAMP pathway; thus, both alpha1A- and alpha1B-ARs couple to Gs, while alpha1D-AR does not. To identify the region responsible for this difference in subtype-specific Gs coupling, we constructed a series of chimeric and a set of point-mutated human alpha1A- and alpha1D-ARs, and examined their signaling ability. Here, we show that the amino acid residues Thr 136 and Val138 in the intracellular loop II of the human alpha1A-AR are intimately involved with Gs coupling.     

Substance P analogs containing alpha,alpha-dialkylated amino acids with potent anticancer activity.

Six analogs (peptides 1-6) of the potent substance P (SP) derivative known as 'Antagonist D' were synthesized by substituting constrained amino acids Aib or Acp (cycloleucine, 1-amino cyclopentane carboxylic acid) at different positions in the Antagonist D sequence: D-Arg(1)-Pro(2)-Lys(3)-Pro(4)-D-Phe(5)-Gln(6)-D-Trp(7)-Phe(8)-D-Trp(9)-Leu(10)-Leu(11)-NH(2). In the preliminary in vitro antiproliferative screening of the analogs on different human cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, peptide 1 was found to be the most active. Further, peptide 1 was butanoylated (analog 5) or octanoylated (analog 6) at the N-terminus. SP analogs 1, 5, and 6 were evaluated in vivo in a xenograft model of human primary colon tumor (PTC) cell line in athymic nude mice and were found to cause tumor regression. This study investigates if the use of the constrained amino acids Aib and Acp in the designed SP analogs can retain the in vitro and in vivo anticancer activities, which could be useful in cancer therapy and drug targeting. Further, the strategy of incorporation of Aib or Acp in biologically active peptides can be exploited in determining the receptor-bound conformation and in transforming these bioactive peptides into pharmacologically useful drugs.     

Peptide design using alpha,beta-dehydro amino acids: from beta-turns to helical hairpins

Incorporation of alpha,beta-dehydrophenylalanine (DeltaPhe) residue in peptides induces folded conformations: beta-turns in short peptides and 3(10)-helices in larger ones. A few exceptions-namely, alpha-helix or flat beta-bend ribbon structures-have also been reported in a few cases. The most favorable conformation of DeltaPhe residues are (phi,psi) approximately (-60 degrees, -30 degrees ), (-60 degrees, 150 degrees ), (80 degrees, 0 degrees ) or their enantiomers. DeltaPhe is an achiral and planar residue. These features have been exploited in designing DeltaPhe zippers and helix-turn-helix motifs. DeltaPhe can be incorporated in both right and left-handed helices. In fact, consecutive occurrence of three or more DeltaPhe amino acids induce left-handed screw sense in peptides containing L-amino acids. Weak interactions involving the DeltaPhe residue play an important role in molecular association. The C--H.O==C hydrogen bond between the DeltaPhe side-chain and backbone carboxyl moiety, pi-pi stacking interactions between DeltaPhe side chains belonging to enantiomeric helices have shown to stabilize folding. The unusual capability of a DeltaPhe ring to form the hub of multicentered interactions namely, a donor in aromatic C--H.pi and C--H.O==C and an acceptor in a CH(3).pi interaction suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures.     

Anticonvulsant activity of cyclopentano amino acids

The hypothesis that certain amino acid analogues possessing a five-membered ring structure or amino acid analogues that can be viewed as fragments derived from such a ring would have anticonvulsant activity was proposed and tested. The compounds 1-aminocyclopentane carboxylic acid, 1-amino-3-methylcyclopentane carboxylic acid, 3-aminotetrahydrothiophene carboxylic acid, and -aminoisobutyric acid were found to protect rats against seizures in the maximal electroshock test but offered no protection against metrazol-(pentylenetetrazol) induced seizures in mice. The structural feature of this class of anticonvulsants that allows for hydrophobic interactions at the receptor site is considered to be a major physical factor necessary in promoting the activity of this class of anticonvulsants.     

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.     

Role of negatively charged amino acids in beta 4 F-loop in activation and desensitization of alpha 3 beta 4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the beta 4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the alpha 3 beta 4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at -40 mV produced inward currents which displayed biphasic desensitization. The EC50 of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC50 increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.     

Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates

Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif that has been proposed to be important for nucleotide sugar binding. To evaluate the importance of the amino acids in this motif, each of the alpha 1,3 FucT motif amino acids was replaced with alanine (alanine scanning mutagenesis) in human FucT VI, and the resulting mutant proteins were analyzed for enzyme activity and kinetically characterized in those cases in which the mutant protein had sufficient activity. Two of the mutant proteins were inactive, six had less than 1% of wild-type activity, and four had approximately 10-50% of wild-type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger Km (5 to 15 times) for GDP-fucose than FucT VI wild-type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a Km at least 10 times larger than wild-type FucT VI for the acceptor substrate, with only a slightly larger (2-3 times) Km for GDP-fucose. Thus mutation of any of the amino acids within the alpha 1,3 FucT motif to Ala affects alpha 1,3-FucT activity, and substitution of Ala for some of the alpha 1,3 FucT motif amino acids results in proteins with altered kinetic constants for both the acceptor and donor substrates. Secondary structure prediction suggests a helix-loop-helix fold for the alpha 1,3 FucT motif, which can be used to rationalize the effects of mutations in terms of 3D structure.