Characterization of dominant negative arrestins that inhibit beta2-adrenergic receptor internalization by distinct mechanisms

Arrestins have been shown to act as adaptor proteins that mediate the interaction of G protein-coupled receptors with the endocytic machinery. In this study, the role of arrestin-3 in receptor internalization was investigated by constructing different arrestin-3 minigenes that could potentially act as dominant negative inhibitors of arrestin function. Expression of arrestin-3 proteins containing amino acids 1-320 or 201-409 resulted in the inhibition of beta2-adrenergic receptor internalization in HEK-293 cells by approximately 40%. Both of these arrestins were diffusely localized within the cytoplasm of transfected cells, were unable to mediate redistribution of receptors to clathrin-coated pits, and did not localize to coated pits in either the presence or absence of receptor and agonist. Arrestin-3(1-320), but not arrestin-3(201-409), bound to light-activated phosphorylated rhodopsin with an affinity comparable with that of wild-type arrestin-3. In contrast, expression of arrestin-3 proteins composed of only the clathrin binding domain, arrestin-3(284-409), and arrestin-3(290-409) resulted in the constitutive localization of these arrestins to coated pits. Arrestin-3(284-409) and arrestin-3(290-409) acted as dominant negative inhibitors of wild-type arrestin function, inhibiting receptor internalization by 70 and 30%, respectively. Carboxyl-terminal deletions of arrestin-3 retained the ability to promote internalization until residues amino-terminal to amino acid 350 were deleted, suggesting that residues in this region also compose part of the clathrin binding domain in addition to the major binding site between residues 371-379. These studies characterize at least two distinct mechanisms, competition for either receptor or clathrin binding, by which dominant negative arrestins inhibit receptor internalization and further define residues within arrestin-3 that constitute the clathrin binding domain.     

Arrestin-independent internalization of the m1, m3, and m4 subtypes of muscarinic cholinergic receptors

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-23689), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the beta2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.     

Inhibition of phospholipase D by clathrin assembly protein 3 (AP3)

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P2 with low affinity. But the PI(4,5)P2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P2. A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.     

Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin

Auxilin was recently identified as cofactor for hsc70 in the uncoating of clathrin-coated vesicles (Ungewickell, E., H. Ungewickell, S.E. Holstein, R. Lindner, K. Prasad, W. Barouch, B. Martin, L.E. Greene, and E. Eisenberg. 1995. Nature (Lond.). 378: 632-635). By constructing different glutathione-S-transferase (GST)-auxilin fragments, we show here that cooperation of auxilin's J domain (segment 813-910) with an adjoining clathrin binding domain (segment 547-814) suffices to dissociate clathrin baskets in the presence of hsc70 and ATP. When the two domains are expressed as separate GST fusion proteins, the cofactor activity is lost, even though both retain their respective functions. The clathrin binding domain binds to triskelia like intact auxilin with a maximum stoichiometry of 3 and concomitantly promotes their assembly into regular baskets. A fragment containing auxilin's J domain associates in an ATP-dependent reaction with hsc70 to form a complex with a half-life of 8 min at 25 degrees C. When the clathrin binding domain and the J domain are recombined via dimerization of their GST moieties, cofactor activity is partially recovered. The interaction between auxilin's J domain and hsc70 causes rapid hydrolysis of bound ATP. Release of inorganic phosphate appears to be correlated with the disintegration of the complex between auxilin's J domain and hsc70. We infer that the metastable complex composed of auxilin, hsc70, ADP, and P(i) contains an activated form of hsc70, primed to engage clathrin that is brought into apposition with it by the DnaJ homologue auxilin.     

Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.     

Arrestin with a single amino acid substitution quenches light-activated rhodopsin in a phosphorylation-independent fashion

Arrestins are members of a superfamily of regulatory proteins that participate in the termination of G protein-mediated signal transduction. In the phototransduction cascade of vertebrate rods, which serves as a prototypical G protein-mediated signaling pathway, the binding of visual arrestin is stimulated by phosphorylation of the C-terminus of photoactivated rhodopsin (Rh*). Arrestin is very selective toward light-activated phosphorhodopsin (P-Rh*). Previously we reported that a single amino acid substitution in arrestin, Arg175Gln, results in a dramatic increase in arrestin binding to Rh* [Gurevich, V. V., & Benovic, J. L. (1995) J. Biol. Chem. 270, 6010-6016]. Here we demonstrate that a similar mutant, arrestin(R175E), binds to light-activated rhodopsin independent of phosphorylation. Arrestin(R175E) binds with high affinity not only to P-Rh* and Rh* but also to light-activated truncated rhodopsin in which the C-terminus phosphorylation sites have been proteolytically removed. In an in vitro assay that monitored rhodopsin-dependent activation of cGMP phosphodiesterase (PDE), wild type arrestin quenched PDE response only when ATP was present to support rhodopsin phosphorylation. In contrast, as little as 30 nM arrestin(R175E) effectively quenched PDE activation in the absence of ATP. Arrestin(R175E) had no effect when the lifetime of Rh* no longer contributed to the time course of PDE activity, suggesting that it disrupts signal transduction at the level of rhodopsin-transducin interaction.     

Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site

Binding of AP180 to clathrin triskelia induces their assembly into 60-70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N-terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C-terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1-20). This protein, localized to synaptic termini, has so far been of unknown function.     

The selectivity of visual arrestin for light-activated phosphorhodopsin is controlled by multiple nonredundant mechanisms

Arrestin plays an important role in quenching phototransduction via its ability to bind to the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Remarkable selectivity of visual arrestin toward this functional form is determined by an elegant sequential multisite binding mechanism. Previous structure-function studies have suggested that the COOH-terminal region of arrestin (residues 356-404) is not directly involved in rhodopsin interaction, but instead plays a regulatory role. This region supports basal arrestin conformation and ensures arrestin's transition into a high affinity rhodopsin-binding state upon an encounter with P-Rh*. Overall, our results corroborate this hypothesis and identify three functional subregions (residues 361-368, 369-378, and 379-404) and individual amino acids involved in the control of arrestin stability and binding selectivity. Two of the most potent mutants, arrestin(1-378) and arrestin(F375A,V376A, F377A) belong to a novel class of constitutively active arrestins with high affinity for P-Rh*, dark P-Rh, and Rh* (but not dark Rh), in contrast to earlier constructed mutants arrestin(R175E) and arrestin(Delta2-16) with high affinity for light-activated forms only. The implications of these findings for the mechanism of arrestin-rhodopsin interaction are discussed in light of the recently determined crystal structure of arrestin.     

Characterization of a highly conserved FAD-binding site in human monoamine oxidase B

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the apoenzyme at Cys397. Our previous studies identified two noncovalent flavin-binding regions in MAO B (residues 6-34 and 39-46) (Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391; Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531). In these regions, Glu34 and Tyr44 were found to be required for the initial binding of FAD. By comparing sequences with enzymes in the oxidoreductase family, we now have found an additional FAD-binding site in MAO B (residues 222-227), which is highly conserved across species (human, bovine, and rat). This conserved sequence contains adjacent glycine and aspartate residues (Gly226 and Asp227). Based on the x-ray crystal structures of several oxidoreductases (Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142; Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764), the Gly residue at the end of a beta-strand facilitates a sharp turn and extends the beta-carbonyl group of Asp to interact with the 3'-hydroxyl group of the ribityl chain of FAD. To assess the hypothesis that Gly226 and Asp227 are involved in FAD binding in MAO B, site-specific mutants that encode substitutions at these positions were prepared and expressed in mammalian COS-7 cells. Our results indicate that Gly226 and the beta-carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD. Our studies also reveal that mutagenesis at Glu34 and Tyr44 not only interferes with covalent flavinylation and catalytic activity of MAO B, but also with noncovalent binding of FAD. Based on these collective results, we propose that the coupling of FAD to the MAO B apoenzyme is a multistep process.     

Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency

Ionic properties of membrane interaction by prothrombin, protein Z, and other vitamin K-dependent proteins were studied to determine the relevance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L. (1997) Biochemistry 36, 5120-5127]. For comparison, multivalent ionic interaction was illustrated by peptides of +3 to +5 net charge and by blood clotting factor V. As expected, the peptides were easily dissociated by salt and gave nominal charge-charge interactions (zazb values) of -13 to -17. Factor V showed much higher binding affinity despite nominal zazb values of about 9. Membrane-bound prothrombin and protein Z showed very low sensitivity to salt as long as calcium was at saturating levels (zazb values of approximately -1.3 to -1.4), appropriate for univalent ionic attraction. Prothrombin contains +3 charge groups (Lys-2, Lys-11, Arg-10) that are absent from the GLA domain (residues 1-35) of protein Z, while protein Z contains -4 charge groups (Gla-11, Asp-34, Asp-35) that are absent in prothrombin. Thus, similar zazb relationships indicated little role for these surface charges in direct membrane contact. Calcium-saturated protein Z bound to phosphatidylcholine (PC) in a manner which indicated the addition of one calcium ion, bringing the total calcium stoichiometry in the protein-membrane complex to at least 8. Protein Z bound to phosphatidic acid (PA) in a manner suggesting the need for a fully ionized phosphate headgroup, a property expected by ion pairing in an isolated environment. Electrostatic calculations showed that the proposed protein site for phosphate interaction was electropositive. The cluster of hydrophobic amino acids (Phe-5, Leu-6, and Val-9) on the surface of prothrombin was electronegative, suggesting a role in the electrostatic architecture of the GLA domain. Overall, membrane binding by vitamin K-dependent proteins appeared consistent with the formation of an ion pair in an isolated environment.     

Charge pair interactions stabilizing ferredoxin-ferredoxin reductase complexes. Identification by complementary site-specific mutations

Ferredoxin reductase (Fd-reductase) supplies electrons to mitochondrial steroid hydroxylase cytochrome P450 enzymes via a [2Fe-2S] ferredoxin. Chemical labeling studies with bovine Fd-reductase have implicated Lys-243 as important in binding to bovine ferredoxin (Hamamoto, I., Kazutaka, K., Tanaka, S., and Ichikawa, Y. (1988) Biochim. Biophys. Acta 953, 207-213). We have used site-directed mutagenesis to examine the role of charged residues in this region of human Fd-reductase in ferredoxin binding. Mutant proteins were expressed in Escherichia coli and were assayed for activity by ferredoxin-mediated electron transfer to cytochrome c. Replacement of Lys-242 (homologous to Lys-243 in bovine Fd-reductase) with Gln and replacement of Arg-241 with Ser had little effect (2.7- and 3.6-fold increased Km, respectively). In contrast, mutants at positions 239 and 243 (R239S and R243Q) exhibited markedly lower affinity for ferredoxin (17.5- and 1,600-fold increased Km, respectively). Studies were also carried out with two ferredoxin charge mutants shown previously to have lowered affinity for Fd-reductase (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Comparisons of the binding of ferredoxin mutants D76N and D79N to Fd-reductase mutants R239S and R243Q suggest that Arg-239 and Arg-243 of Fd-reductase each interact directly with both Asp-76 and Asp-79 of ferredoxin during formation of the complex between the two proteins. These results support the hypothesis that specific electrostatic interactions involving this region are important in stabilizing the ferredoxin-Fd-reductase complex.     

Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state

Arrestin plays an important role in quenching phototransduction via its ability to interact specifically with the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Previous studies have demonstrated that Arg175 in bovine arrestin is directly involved in the phosphorylation-dependent binding of arrestin to rhodopsin and seems to function as a phosphorylation-sensitive trigger. In this study, we further probed the molecular mechanism of phosphorylation recognition by substituting 19 different amino acids for Arg175. We also assessed the effects of mutagenesis of several other highly conserved residues within the phosphorylation-recognition region (Val170, Leu172, Leu173, Ile174, Val177, and Gln178). The binding of all of these mutants to P-Rh*, light-activated rhodopsin, and truncated rhodopsin, which lacks the carboxyl-terminal phosphorylation sites, was then characterized. Overall, our results suggest that arrestin interaction with the phosphorylated carboxyl-terminal domain of rhodopsin activates two relatively independent changes in arrestin: (a) mobilization of additional binding sites and (b) increased affinity of the phosphorylation-recognition region for the rhodopsin carboxyl-terminal domain. Together, these two mechanisms ensure the exquisite selectivity of arrestin toward P-Rh*. Mutagenesis of residues that play a major role in binding site mobilization and phosphorylation-recognition enabled us to create "constitutively active" (phosphorylation-independent) arrestin mutants that have high affinity for both P-Rh* and light-activated rhodopsin. The introduction of a negative charge in position 175 was particularly effective in this respect. A detailed molecular model of phosphorylation-recognition is proposed.     

Site-directed mutagenesis of the yeast V-ATPase B subunit (Vma2p)

The B subunit of the vacuolar (H+)-ATPase (V-ATPase) has previously been shown to participate in nucleotide binding and to possess significant sequence homology with the alpha subunit of the mitochondrial F-ATPase, which forms the major portion of the noncatalytic nucleotide binding sites and contributes several residues to the catalytic sites of this complex. Based upon the recent x-ray structure of the mitochondrial F1 ATPase (Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker, J.E. (1994) Nature 370,621-628), site-directed mutagenesis of the yeast VMA2 gene has been carried out in a strain containing a deletion of this gene. VMA2 encodes the yeast V-ATPase B subunit (Vma2p). Mutations at two residues postulated to be contributed by Vma2p to the catalytic site (R381S and Y352S) resulted in a complete loss of ATPase activity and proton transport, with the former having a partial effect on V-ATPase assembly. Interestingly, substitution of Phe for Tyr-352 had only minor effects on activity (15-30% inhibition), suggesting the requirement for an aromatic ring at this position. Alteration of Tyr-370, which is postulated to be near the adenine binding pocket at the noncatalytic sites, to Arg, Phe, or Ser caused a 30-50% inhibition of proton transport and ATPase activity, suggesting that an aromatic ring is not essential at this position. Finally, mutagenesis of residues in the region corresponding to the P-loop of the alpha subunit (H180K, H180G, H180D, N181V) also inhibited proton transport and ATPase activity by approximately 30-50%. None of the mutations in either the putative adenine binding pocket nor the P-loop region had any effect on the ability of Vma2p to correctly fold nor on the V-ATPase to correctly assemble. The significance of these results for the structure and function of the nucleotide binding sites on the B subunit is discussed.     

Crystal structure of the complex between VEGF and a receptor-blocking peptide

Vascular endothelial growth factor (VEGF) is a specific and potent angiogenic factor and, therefore, a prime therapeutic target for the development of antagonists for the treatment of cancer. As a first step toward this goal, phage display was used to generate peptides that bind to the receptor-binding domain (residues 8-109) of VEGF and compete with receptor [Fairbrother, W. J., Christinger, H. W., Cochran, A. G., Fuh, G., Keenan, C. J., Quan, C., Shriver, S. K., Tom, J. Y. K., Wells, J. A., and Cunningham, B. C. (1999) Biochemistry 38, 17754-17764]. The crystal structure of VEGF in complex with one of these peptides was solved and refined to a resolution of 1.9 A. The 20-mer peptide is unstructured in solution and adopts a largely extended conformation when bound to VEGF. Residues 3-8 form a beta-strand which pairs with strand beta6 of VEGF via six hydrogen bonds. The C-terminal four residues of the peptide point away from the growth factor, consistent with NMR data indicating that these residues are flexible in the complex in solution. In contrast, shortening the N-terminus of the peptide leads to decreased binding affinities. Truncation studies show that the peptide can be reduced to 14 residues with only moderate effect on binding affinity. However, because of the extended conformation and the scarcity of specific side-chain interactions with VEGF, the peptide is not a promising lead for small-molecule development. The interface between the peptide and VEGF contains a subset of the residues recognized by a neutralizing Fab fragment and overlaps partially with the binding site for the Flt-1 receptor. The location of the peptide-binding site and the hydrophilic character of the interactions with VEGF resemble more the binding mode of the Fab fragment than that of the receptor.     

Residue lysine-34 in GroES modulates allosteric transitions in GroEL

The conserved residue Lys-34 in GroES was replaced by alanine and glutamic acid using site-directed mutagenesis. This residue is near the carboxy terminus of the mobile loop in GroES (residues 17-32) which becomes immobilized upon formation of the GroEL/GroES complex [Landry et al. (1993) Nature 364, 255-258]. Both charge neutralization (Lys-34-->Ala) and charge reversal (Lys-34-->Glu) at this position have little effect on the binding constant of GroES to GroEL, but they increase the enhancement by GroES of cooperativity in ATP hydrolysis by GroEL. This is reflected by a change in the Hill coefficient (at 10 mM K+) from 4.10 (+/- 0.22) in the presence of wild-type GroES to 5.17 (+/- 0.24) and 4.46 (+/- 0.14) in the presence of the GroES mutants Lys-34-->Ala and Lys-34-->Glu, respectively. The results are interpreted using the Monod-Wyman-Changeux (MWC) model for cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118]. They suggest that Lys-34 in GroES modulates the allosteric transition in GroEL by stabilizing a relaxed (R)-like state.     

Agonist-receptor-arrestin, an alternative ternary complex with high agonist affinity

The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the beta2-adrenergic receptor (beta2AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of beta2AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated beta2AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonist-receptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization.     

Diversity and properties of calcium channel types in NG108-15 hybrid cells

Several lysines (Lys) were determined to be involved in the regulation of the ADP-glucose (Glc) pyrophosphorylase from spinach leaf and the cyanobacterium Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711; Y. Charng, A.A. Iglesias, J. Preiss [1994] J Biol Chem 269: 24107-24113). Site-directed mutagenesis was used to investigate the relative roles of the conserved Lys in the heterotetrameric enzyme from potato (Solanum tuberosum L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9- and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12- and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg2+. These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.     

Regulation of the p85/p110alpha phosphatidylinositol 3'-kinase. Distinct roles for the n-terminal and c-terminal SH2 domains

Our previous studies on the p85/p110alpha phosphatidylinositol 3-kinase showed that the p85 regulatory subunit inhibits the p110alpha catalytic subunit, and that phosphopeptide activation of p85/p110alpha dimers reflects a disinhibition of p110alpha (Yu, J., Zhang, Y., McIlroy, J., Rordorf-Nikolic, T., Orr, G. A., and Backer, J. M. (1998) Mol. Cell. Biol. 18, 1379-1387). We now define the domains of p85 required for inhibition of p110alpha. The iSH2 domain of p85 is sufficient to bind p110alpha but does not inhibit it. Inhibition of p110alpha requires the presence of the nSH2 domain linked to the iSH2 domain. Phosphopeptides increase the activity of nSH2/iSH2-p110alpha dimers, demonstrating that the nSH2 domain mediates both inhibition of p110alpha and disinhibition by phosphopeptides. In contrast, phosphopeptides did not increase the activity of iSH2/cSH2-p110alpha dimers, or dimers composed of p110alpha and an nSH2/iSH2/cSH2 construct containing a mutant nSH2 domain. Phosphopeptide binding to the cSH2 domain increased p110alpha activity only in the context of an intact p85 containing both the nSH2 domain and residues 1-322 (the SH3, proline-rich and breakpoint cluster region-homolgy domains). These data suggest that the nSH2 domain of p85 is a direct regulator of p110alpha activity. Regulation of p110alpha by phosphopeptide binding to the cSH2 domain occurs by a mechanism that requires the additional presence of the nSH2 domain and residues 1-322 of p85.     

Dimerization/docking domain of the type Ialpha regulatory subunit of cAMP-dependent protein kinase. Requirements for dimerization and docking are distinct but overlapping

Based on increasing evidence that the type I R subunits as well as the type II R subunits localize to specific subcellular sites, we have carried out an extensive characterization of the stable dimerization domain at the N terminus of RIalpha. Deletion mutants as well as alanine scanning mutagenesis were used to delineate critical regions as well as particular amino acids that are required for homodimerization. A set of nested deletion mutants defined a minimum core required for dimerization. Two single site mutations on the C37H template, RIalpha(F47A) and RIalpha(F52A), were sufficient to abolish dimerization. In addition to serving as a dimerization motif, this domain also serves as a docking surface for binding to dual specificity anchoring proteins (D-AKAPs) (Huang, L. J., Durick, K., Weiner, J. A., Chun, J., and Taylor, S. S. (1997) J. Biol. Chem. 272, 8057-8064; Huang, L. J., Durick, K., Weiner, J. A., Chun, J., and Taylor, S. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11184-11189). A similar strategy was used to map the sequence requirements for anchoring of RIalpha to D-AKAP1. Although dimerization appears to be essential for anchoring to D-AKAP1, anchoring can also be abolished by the following single site mutations: C37H, V20A, and I25A. These sites define "hot spots" for the anchoring surface since each of these dimeric proteins are deficient in binding to D-AKAP1. In contrast to earlier predictions, the alignment of the dimerization/docking domains of RIalpha and RII show striking similarities yet subtle differences not only in their secondary structure (Newlon, M. G., Roy, M., Hausken, Z. E., Scott, J. D., and Jennings. P. A. (1997) J. Biol. Chem. 272, 23637-23644) but also in the distribution of residues important for both docking and dimerization functions.     

Residues 21-30 within the extracellular N-terminal region of the C5a receptor represent a binding domain for the C5a anaphylatoxin

The functions of the C5a anaphylatoxin are expressed through its interaction with a cell-surface receptor with seven transmembrane helices. The interaction of C5a with the receptor has been explained by a two-site model whereby recognition and effector sites on C5a bind, respectively, to recognition and effector domains on the receptor, leading to receptor activation (Chenoweth, D. E., and Hugli, T. E. (1980) Mol. Immunol. 17, 151-161. In addition, the extracellular N-terminal region of the C5a receptor has been implicated as the recognition domain for C5a, responsible for approximately 50% of the binding energy of the C5a-receptor complex (Mery, L., and Boulay, F. (1994) J. Biol. Chem. 269, 3457-3463; DeMartino, J. A., Van Riper, G., Siciliano, S. J., Molineaux, C. J., Konteatis, Z. D., Rosen, H., and Springer, M. S. (1994) J. Biol. Chem. 269, 14446-14450). In this work, the interactions of C5a with the N-terminal domain of the C5a receptor were examined by use of recombinant human C5a molecules and peptide fragments M1NSFN5YTTPD10YGHYD15DKDTL20DLNTP25VDKTS30NTLR(hC5aRF-1-34), acetyl-HYD15DKDTL20DLNTP25VDKTS30NTLR (hC5aRF-13-34), and acetyl-TL20DLNTP25VDKTS30N-amide (hC5aRF-19-31) derived from human C5a receptor. Binding induced resonance perturbations in the NMR spectra of the receptor fragments and the C5a molecules indicated that the isolated Nterminal domain or residues 1-34 of the C5a receptor retain specific binding to C5a and to a C5a analog devoid of the agonistic C-terminal tail in the intact C5a. Residues of C5a perturbed by the binding of the receptor peptides are localized within the helical core of the C5a structure, in agreement with the results from functional studies employing mutated C5a and intact receptor molecules. All three receptor peptides, hC5aRF-1-34, hC5aRF-13-34, and hC5aRF-19-31, responded to the binding of C5a through the 21-30 region containing either hydrophobic, polar, or positively charged residues such as Thr24, Pro25, Val26, Lys28, Thr29, and Ser30. The 21-30 segment of all three receptor fragments was found to have a partially folded conformation in solution, independent of residues 1-18. These results indicate that a short peptide sequence, or residues 21-30, of the C5a receptor N terminus may constitute the binding domain for the recognition site on C5a.