Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin

BACKGROUND: The third cytoplasmic loop of rhodopsin (Rho EF) is important in signal transduction from the retinal in rhodopsin to its G protein, transducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from light-activated phosphorylated rhodopsin. RESULTS: We replaced eight residues of the EF loop of bacteriorhodopsin (BR) with 24 residues from the third cytoplasmic loop of bovine Rho EF. The surfaces of purple membrane containing the mutant BR (called IIIN) were imaged by atomic force microscopy (AFM) under physiological conditions to a resolution of 0.5-0.7 nm. The crystallinity and extracellular surface of IIIN were not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF were excised by V8 protease, revealing helices E and F in the AFM topographs. Rho EF was modeled onto the BR structure, and the envelope derived from the AFM data of IIIN was used to select probable models. CONCLUSIONS: A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.     

Stabilization of iron-sulfur cluster F(X) by intra-subunit interactions unraveled by suppressor and second site-directed mutations in PsaB of Photosystem I

Intra-subunit interactions in the environment of the iron-sulfur cluster F(X) in Photosystem I (PS I) of Synechocystis sp. PCC 6803 were studied by site-directed and second site suppressor mutations. In subunit PsaB, the cysteine ligand (C565) of F(X) and a conserved aspartate (D566) adjacent to C565 were modified. The resulting mutants D566E, C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I in the thylakoids of the cyanobacterium. Yet, this is the first report of cells of the second site-suppressor mutant (D566E/L416P) and of second site-directed mutant (C565S/D566E) in PsaB that could grow autotrophically in light and were found to assemble a stable functional PS I containing all three iron-sulfur centers, F(X) and F(A/B). The newly resolved structure of PS I (PDB 1JB0) was used to interpret the functional interactions among the amino acid residues. It is suggested that the stability of F(X) is supported by a salt bridge formed between D566, which is adjacent to the cysteine ligand C565 of the iron-sulfur cluster located on loop hi, and R703 located at the start of loop jk. Hydrogen bond between R703 and D571 at the start of loop hi further stabilizes the arginine. Lengthening of the side by 1.2 A chain in mutation D566E caused destabilization of F(X). The extended side-chain was compensated for by the Fe-O, which is 0.3 A shorter than the Fe-S bond resulting in stabilization of the F(X) in the double mutations C565S/D566E. The suppressor mutation D566E/L416P allowed greater freedom for the salt bridge E566-R703, thus relieving the pressure introduced by the D566E replacement and enabling the formation of F(X). F(X) and R703 are therefore stabilized through short- and long-range interactions of the inter-helical loops between h-i, j-k and f-g, respectively.     

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189–209) and loop B (res. 577–608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.     

Thermostabilization of Pichia stipitis xylitol dehydrogenase by mutation of structural zinc-binding loop

Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 degrees C higher thermal transition temperature (T(CD)) and 20.8 degrees C higher half denaturation temperature (T(1/2)) compared with wild-type.     

The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors

The competitive antagonist d-tubocurarine (curare) has greater potency at mouse than at human 5-hydroxytryptamine 3A (5-HT3A) receptors, despite 84% amino acid sequence identity between the receptors. Within the ligand binding domain of this receptor are six loops (A-F). A previous report demonstrated that loop C of the 5-HT3A receptor contributed to differential potency between the receptors [Hope, A. G. et al. (1999) Mol. Pharmacol. 55, 1037-1043]. The present study tested the hypothesis that loop F plays a significant role in conferring interspecies curare potency differences. Wild-type, chimeric, and point mutant 5-HT3A receptors were expressed in Xenopus oocytes, and two-electrode voltage clamp electrophysiological recordings were performed. Our data suggest that loops C and F contribute to curare potency, given that the curare IC50's (concentration of drug that produces 50% inhibition of the response) for chimeric human receptors with substitutions of mouse residues in loop C (40.07 +/- 2.52 nM) or loop F (131.8 +/- 5.95 nM) were intermediate between those for the mouse (12.99 +/- 0.77 nM) and human (1817 +/- 92.36 nM) wild-type receptors. Two human point mutant receptors containing mouse receptor substitutions in loop F (H-K195E or H-V202I) had significantly lower curare IC50's than that of the human receptor. The human double mutant receptor, H-K195E,V202I, had the same curare IC50 (133.8 +/- 6.38 nM) as that of the human receptor containing all six loop F mouse substitutions. These results demonstrate that two loop F residues make a significant contribution in determining curare potency at the 5-HT3A receptor.     

Structural interactions between FXYD proteins and Na+,K+-ATPase: alpha/beta/FXYD subunit stoichiometry and cross-linking

Interactions of rat FXYD4 (corticosteroid hormone-induced factor (CHIF)), FXYD2 (gamma), or FXYD1 (phospholemman (PLM)) proteins with rat alpha1 subunits of Na(+),K(+)-ATPase have been analyzed by co-immunoprecipitation and covalent cross-linking. In detergent-solubilized membranes from HeLa cells expressing both gamma and CHIF or CHIF and hemagglutinin A-tagged CHIF, mixed complexes of CHIF and gamma or CHIF and hemagglutinin A-tagged CHIF with alpha/beta subunits are undetectable. This implies that the alpha/beta/FXYD protomer is the major species in detergent solution. A lipid-soluble cysteine-cysteine bifunctional reagent, dibromobimane, cross-links CHIF to alpha in colonic membranes but not gamma or PLM to alpha in kidney or heart membranes, respectively. Sequence comparisons of the FXYD proteins suggested that Cys-49 in the trans-membrane segment of CHIF could be involved. In detergent-solubilized HeLa cell membranes, dibromobimane cross-links wild-type CHIF to alpha but not the C49F mutant, and also the corresponding F36C mutant but not wild-type gammab, and F48C but not wild-type PLM. C140S, C338A, C804A, and C966S mutants of the alpha subunit have been expressed. Only the C140S mutant prevents cross-linking with CHIF. The data demonstrated the proximity of trans-membrane segments of CHIF, gamma, and PLM to M2 of alpha. Molecular modeling is consistent with location of the trans-membrane segment of all FXYD proteins between M2, M6, and M9 and the proximity of Cys-49 of CHIF or Phe-36 of gamma with Cys-140 of M2. Cross-linking also demonstrated CHIF-alpha and CHIF-beta proximities in extra-membrane regions, similar to the evidence for gamma-alpha and gamma-beta cross-links.     

Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase.

Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Delta p, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III. With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues beta A398, beta S404, beta I406, beta G408, beta M409 and beta V411 in loop D, and residue beta Y431 in loop E were selected. In addition, the previously made mutants betaD392C and betaT393C in loop D, and beta G430C and beta A432C in loop E, were included. All loop D and E mutants, especially beta I406C and beta G430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild-type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.     

Second-site suppressor mutations for the serine 202 to phenylalanine substitution within the interdomain loop of the tetracycline efflux protein Tet(C)

The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly reduces tetracycline resistance and efflux activity (Saraceni-Richards, C. A., and Levy, S. B. (2000) J. Biol. Chem. 275, 6101-6106). Second-site suppressor mutations were identified following hydroxylamine and nitrosoguanidine mutagenesis. Three mutations, L11F in transmembrane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8-9, restored a wild type level of resistance and an active efflux activity in Escherichia coli cells bearing the mutant tet(C) gene. The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mutant, whereas L11F and A213T Tet(C) protein mutants were overexpressed. Introduction of each single mutation into the wild type tet(C) gene by site-directed mutagenesis did not alter tetracycline resistance or efflux activity. These secondary mutations may restore resistance by promoting a conformational change in the protein to accommodate the S202F mutation. The data demonstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance.     

A flexible loop in tyrosine hydroxylase controls coupling of amino acid hydroxylation to tetrahydropterin oxidation

The role of a polypeptide loop in tyrosine hydroxylase (TyrH) whose homolog in phenylalanine hydroxylase (PheH) takes on a different conformation when substrates are bound has been studied using site-directed mutagenesis. The loop spans positions 177 to 191; alanine was introduced into those positions, introducing one alanine substitution per TyrH variant. Mutagenesis of residues in the center of the loop resulted in alterations in the KM values for substrates, the Vmax value for dihydroxyphenylalanine (DOPA) synthesis, and the coupling of tetrahydropterin oxidation to tyrosine hydroxylation. The variant with the most altered KM value for 6-methyltetrahydropterin was TyrH F184A. The variants with the most affected K(tyr) values were those with substitutions in the center of the loop, TyrH K183A, F184A, D185A, P186A and D187A. These five variants also had the most reduced Vmax values for DOPA synthesis. Alanine substitution in positions 182-186 resulted in lowered ratios of tyrosine hydroxylation to tetrahydropterin oxidation. TyrH F184Y and PheH Y138F, variants with the residue at the center of the loop substituted with the residue present at the homologous position in the other hydroxylase, were also studied. The V/K(tyr) to V/K(phe) ratios for these variants were altered significantly, but the results did not suggest that F184 of TyrH or Y138 of PheH plays a dominant role in determining amino acid substrate specificity.     

Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.     

Cytoplasmic surface structures of bacteriorhodopsin modified by site-directed mutations and cation binding as revealed by <Superscript>13</Superscript>C NMR

We have examined how cytoplasmic surface structures of [3-(13)C]Ala-labeled bacteriorhodopsin (bR), consisting of the C-terminal alpha-helix and cytoplasmic loops, are altered by site-directed mutations at the former (R227Q) and the latter (A160G, E166G, and A168G) and by cation binding, by means of displacements of the (13)C NMR peaks of Ala228 and Ala233 (C-terminal alpha-helix), Ala103 (C-D loop), and Ala160 (E-F loop). Cytoplasmic ends of the B and F helices were found to undergo fluctuation motions on the order of 10(-5) s, when such surface structures were disrupted, as viewed from suppressed (13)C NMR signals. This happens also for deionized blue membranes of wild type and A160G, with accelerated fluctuations in the loops. Further, cytoplasmic surface structures of Na(+)-regenerated purple membrane from the blue membrane were significantly modified by Ca(2+) ions up to 1 mM under relatively low ionic strength of 10 mM NaCl, although they are very similar at high ionic strength (100 mM NaCl). To interpret these findings, the following two surface structures were proposed. The C-terminal alpha-helix of the wild type at ambient temperature is involved in a perturbed type, probably tilted toward the direction of the B and F helices, to prevent unnecessary fluctuations of these helices for efficient proton uptake during the photocycle. An unperturbed type of helix is achieved when such a surface structure was disrupted at low temperature or in an M-like state. This view is consistent with previously published data for the "proton binding cluster" consisting of Asp104, Glu166, and Glu234.     

Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin.

Structural requirements for the activation of transducin by rhodopsin have been studied by site-specific mutagenesis of bovine rhodopsin. A variety of single amino acid replacements and amino acid insertions and deletions of varying sizes were carried out in the two cytoplasmic loops CD (amino acids 134-151) and EF (amino acids 231-252). Except for deletion mutant delta 137-150, all the mutants bound 11-cis-retinal and displayed normal spectral characteristics. Deletion mutant delta 236-239 in loop EF caused a 50% reduction of transducin activation, whereas deletion mutant delta 244-249 and the larger deletions in loop EF abolished transducin activation. An 8-amino acid deletion in the cytoplasmic loop CD as well as a replacement of 13 amino acids with an unrelated sequence showed no transducin activation. Several single amino acid substitutions also caused significant reduction in transducin activation. The conserved charged pair Glu-134/Arg-135 in the cytoplasmic loop CD was required for transducin activation; its reversal or neutralization abolished transducin activation. Three amino acid replacements in loop EF (S240A, T243V, and K248L) resulted in significant reduction in transducin activation. We conclude that 1) both the cytoplasmic loops CD and EF are required for transducin activation, and 2) effective functional interaction between rhodopsin and transducin involves relatively large peptide sequences in the cytoplasmic loops.     

Evidence for interactions between helices 5 and 8 and a role for the interdomain loop in tetracycline resistance mediated by hybrid Tet proteins

An interdomain hybrid Tet protein consisting of a class C alpha domain and a class B beta domain (Tet(C/B)) lacks detectable efflux ability and provides only minimal levels of resistance to tetracycline (Tc) (3 microg/ml) compared with intact class B (256 microg/ml) and class C (64 microg/ml). Twenty-one independently isolated mutants of the Tet(C/B) protein with increased Tc resistance were generated by random chemical mutagenesis. Nine mutants with a Glu substitution for Gly-152 in helix 5 of the class C alpha domain produced a resistance of 48 microg/ml, whereas another 9 with an Asp replacement of Gly-247 in helix 8 of the class B beta domain mediated resistance at 32 microg/ml. The third type of mutation, found in 3 mutants expressing 24 microg/ml resistance, was a S202F replacement in the putative interdomain cytoplasmic loop of Tet(C/B). The latter underscores a previously unappreciated function of the interdomain cytoplasmic loop. All three types of Tet(C/B) mutant proteins were expressed in amounts comparable with that of the original protein and demonstrated restored energy-dependent efflux of tetracycline. Site-directed mutational analysis demonstrated that a Gly-247 to Asn mutation could also facilitate Tc resistance by the Tet(C/B) hybrid, and a negatively charged side chain at position 152 was required for Tet(C/B) activity. These mutations appear to promote the necessary functional interactions between the interclass domains that do not occur in the Tet(C/B) hybrid protein and suggest a direct association between helix 5 and helix 8 in the function of Tet efflux proteins.     

Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.     

Exploring the mechanism of zanamivir resistance in a neuraminidase mutant: a molecular dynamics study

It is critical to understand the molecular basis of the drug resistance of influenza viruses to efficiently treat this infectious disease. Recently, H1N1 strains of influenza A carrying a mutation of Q136K in neuraminidase were found. The new strain showed a strong Zanamivir neutralization effect. In this study, normal molecular dynamics simulations and metadynamics simulations were employed to explore the mechanism of Zanamivir resistance. The wild-type neuraminidase contained a 3(10) helix before the 150 loop, and there was interaction between the 150 and 430 loops. However, the helix and the interaction between the two loops were disturbed in the mutant protein due to interaction between K136 and nearby residues. Hydrogen-bond network analysis showed weakened interaction between the Zanamivir drug and E276/D151 on account of the electrostatic interaction between K136 and D151. Metadynamics simulations showed that the free energy landscape was different in the mutant than in the wild-type neuraminidase. Conformation with the global minimum of free energy for the mutant protein was different from the wild-type conformation. While the drug fit completely into the active site of the wild-type neuraminidase, it did not match the active site of the mutant variant. This study indicates that the altered hydrogen-bond network and the deformation of the 150 loop are the key factors in development of Zanamivir resistance. Furthermore, the Q136K mutation has a variable effect on conformation of different N1 variants, with conformation of the 1918 N1 variant being more profoundly affected than that of the other N1 variants studied in this paper. This observation warrants further experimental investigation.     

Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II

The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.     

The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide

Sequence and predicted structural similarities between members of the Cys loop superfamily of ligand-gated ion channel receptors and the acetylcholine binding protein (AChBP) suggest that the ligand-binding site is formed by six loops that intersect at subunit interfaces. We employed site-directed mutagenesis to investigate the role of amino acids from the loop C region of the murine 5-HT(3AS)R in interacting with two structurally different agonists, serotonin (5-HT) and m-chlorophenylbiguanide (mCPBG). Mutant receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies. Electrophysiological assays were employed to identify changes in response characteristics and relative efficacies of mCPBG and the partial agonist, 2-methyl 5-HT (2-Me5-HT). We have also constructed novel 5-HT and mCPBG docked models of the receptor binding site based on homology models of the AChBP. Both ligand-docked models correlate well with results from mutagenesis and electrophysiological assays. Four key amino acids were identified as being important to ligand binding and/or gating of the receptor. Among these, I228 and D229 are specific for effects mediated by 5-HT compared to mCPBG, indicating a differential interaction of these ligands with loop C. Residues F226 and Y234 are important for both 5-HT and mCPBG interactions. Mutations at F226, I228, and Y234 also altered the relative efficacies of agonists, suggesting a role in the gating mechanism.     

Bifunctional peptides derived from homologous loop regions in the laminin alpha chain LG4 modules interact with both alpha 2 beta 1 integrin and syndecan-2

Laminin alpha chains show diverse biological functions in a chain-specific fashion. The laminin G-like modules (LG modules) of the laminin alpha chains consist of a 14-stranded beta-sheet sandwich structure with biologically active sequences found in the connecting loops. Previously, we reported that connecting loop regions between beta-strands E and F in the mouse laminin alpha chain LG4 modules exhibited chain-specific activities. In this study, we focus on the homologous loop regions in human laminin alpha chain LG4 modules using five synthetic peptides (hEF-1-hEF-5). These homologous peptides induced chain-specific cellular responses in various cell types. Next, to examine the dual-receptor recognition model, we synthesized chimeras (cEF13A-cEF13E) derived from peptides hEF-1 and hEF-3. All of the chimeric peptides promoted fibroblast attachment as well as the parental peptides. Attachment of fibroblasts to cEF13A and cEF13B was inhibited by anti-integrin alpha2 and beta1 antibodies and by heparin, while cell adhesion to cEF13C, cEF13D, and cEF13E was blocked only by heparin. Actin organization of fibroblasts on cEF13C was not different from that on hEF-3, but cEF13B induced membrane ruffling at the tips of the actin stress fibers. These results suggest that cEF13B had bifunctional effects on cellular behaviors through alpha2beta1 integrin and heparin/heparan sulfate proteoglycan. We conclude that the approach utilizing chimeric peptides is useful for examining cellular mechanisms in dual-receptor systems.     

Molecular basis of pH and Ca2+ regulation of aquaporin water permeability

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca(2+) increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can "tune" the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca(2+) sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca(2+) and pH modulation are separable and arise from processes on opposite sides of the membrane.