Regulation of G proteins by human 5-HT1a receptor TM3/i2 and TM5/i3 loop peptides

A bioactive synthetic 11 amino acid peptide probe (P11) was constructed according to the published sequence of the human 5HT1a receptor. The probe was used to enhance understanding of cytoplasmic loop 2/G protein coupling and activation. Additionally, two peptides (P8, P9) from the cytoplasmic loop 3 region were synthesized and studied. These probes were tested in a model system of human 5HT1a receptor stably expressed in Chinese Hamster Ovary cells. In agonist inhibition studies, P11 was active in all three receptor preparations tested: whole cells, membrane bound, and solubilized. In analyses of the membrane bound receptor system, P11 demonstrated uncompetitive inhibition characteristics. When forskolin-stimulated cAMP levels were measured, P11 was inactive in this negatively coupled system. Utilizing a [35S]gamma-S-GTP incorporation assay, P11 was unable to stimulate G protein incorporation of GTP. While P8 and P9 were also broadly active as non-competitive agonist inhibitors, their characteristics differed in the signal transduction system. P8 and P9 did not significantly change forskolin-stimulated cAMP levels. However, P8 increased [35S]gamma-S-GTP incorporation, while P9 decreased incorporation. Thus, P11, a synthetic peptide from the TM3/i2 region of the receptor, provides suggestive evidence that this receptor region is involved in G protein coupling but not activation. On the other hand, P8 and P9 activities suggest that the TM5/i3 region is involved in both coupling to and regulation of G protein activity. The current evidence from these cytoplasmic loop regions is discussed in the overall context of an emerging model for human 5HT1a receptor-G protein interactions.     

A G protein gamma subunit-specific peptide inhibits muscarinic receptor signaling

Muscarinic acetylcholine receptors modulate the function of a variety of effectors through heterotrimeric G proteins. A prenylated peptide specific to the G protein gamma5 subunit type inhibits G protein activation by the M2 muscarinic receptor in a reconstitution assay. Scrambling the amino acid sequence of the peptide significantly reduces the efficacy of the peptide. The peptide does not disrupt the G protein heterotrimer. In cultured sympathetic neurons, the gamma5 peptide inhibits modulation of Ca(2+) current by the M4 receptor. Peptide activity is specific, the scrambled peptide and peptides specific to two other members of the G protein gamma subunit family are significantly less effective. The gamma5 peptide has no effect on Ca(2+) current modulation by the alpha2-adrenergic and somatostatin receptors. In addition, the gamma5 peptide inhibits muscarinic receptor signaling in spinal cord slices with specificity. These results support a specific role for G protein gamma subunit types in signal transduction, most likely at the receptor-G protein interface.     

A highly effective dominant negative alpha s construct containing mutations that affect distinct functions inhibits multiple Gs-coupled receptor signaling pathways

To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative alpha(s) mutant containing substitutions that alter distinct functions was produced and tested for its effects on G(s)-coupled receptor activity in HEK-293 cells. Mutations in the alpha3beta5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for betagamma, and A366S, which decreases affinity for GDP. This triple alpha(s) mutant can inhibit signaling to G(s) from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this alpha(s) mutant blocks all signaling from the calcitonin receptor to G(q). These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein alpha subunits can compete with each other for binding to the same receptor. This dominant negative alpha(s) construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.     

G alpha minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation

The C termini of G protein alpha subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a G alpha(i) peptide to bind the A(1) adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998 ) J. Biol. Chem. 273, 14912--14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of G alpha(i) could specifically inhibit downstream responses to agonist stimulation of the muscarinic M(2) receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610--6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of G alpha subunits. Minigene vectors expressing G alpha C-terminal peptides (G alpha(i), G alpha(q), G alpha(12), and G alpha(13)) or the control minigene vector, which expresses the G alpha(i) peptide in random order (G(iR)), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal G alpha minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.     

Real-time analysis of G protein-coupled receptor reconstitution in a solubilized system

Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of the N-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Galpha subunits premixed with bovine brain Gbetagamma subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and Galpha(i3) protein interacted with an affinity of approximately 10(-6) m with other alpha subunits exhibiting lower affinities (Galpha(i3) > Galpha(i2) > Galpha(i1) Galpha(o)). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Galpha(i) C-terminal regions, by Galpha(i) mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes.     

G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.     

Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells

To determine the intracellular signaling mechanism of the 5-HT(2C) receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta2 adrenergic receptor-mediated activation of cAMP but not 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCbeta1M, mimicking the domain of phospholipase Cbeta1 (PLCbeta1) interacting with active Galpha(q), also blocked 5-HT(2C) receptor activation. In contrast, peptides PLCbeta2M and Phos that bind to and sequester free Gbetagamma subunits were ineffective at blocking 5-HT(2C) receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gbetagamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Galpha(q) subunits mediate endogenous 5-HT(2C) receptor activation of PLCbeta and that Gbetagamma subunits released from Galpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.     

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. II. Elucidation of G protein specificity determinants

Within any given cell many G protein-coupled receptors are expressed in the presence of multiple G proteins, yet most receptors couple to a specific subset of G proteins to elicit their programmed response. Numerous studies demonstrate that the carboxyl-terminal five amino acids of the Galpha subunits are a major determinant of specificity, however the receptor determinants of specificity are less clear. We have used a collection of 133 functional mutants of the C5a receptor obtained in a mutagenesis screen targeting the intracellular loops and the carboxyl terminus (Matsumoto, M. L., Narzinski, K., Kiser, P. D., Nikiforovich, G. V., and Baranski, T. J. (2007) J. Biol. Chem. 282, 3105-3121) to investigate how specificity is encoded. Each mutant, originally selected for its ability to signal through a nearly full-length Galpha(i) in yeast, was tested to see whether it could activate three versions of chimeric Galpha subunits consisting of Gpa1 fused to the carboxyl-terminal five amino acids of Galpha(i), Galpha(q), or Galpha(s) in yeast. Surprisingly the carboxyl-terminal tail of the C5a receptor is the most important specificity determinant in that nearly all mutants in this region showed a gain in coupling to Galpha(q) and/or Galpha(s). More than half of the receptors mutated in the second intracellular loop also demonstrated broadened G protein coupling. Given a lack of selective advantage for this broadened signaling in the initial screen, we propose a model in which the carboxyl-terminal tail acts together with the intracellular loops to generate a specificity filter for receptor-G protein interactions that functions primarily to restrict access of incorrect G proteins to the receptor.     

Immunoprecipitation of alpha 2a-adrenergic receptor-GTP-binding protein complexes using GTP-binding protein selective antisera. Changes in receptor/GTP-binding protein interaction following agonist binding.

The nature of the interaction of cloned alpha 2a-adrenergic receptors from LLC-PK1-O clone cells with G proteins was investigated using an immunoprecipitation approach. Following solubilization of the alpha 2a receptors, antiserum 8730, which is directed against the C-terminal region of Gi alpha, immunoprecipitated alpha 2a receptor-Gi alpha complexes. The immunoprecipitation was specific since it could be blocked by the peptide to which antiserum 8730 was generated. Antisera 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3) immunoprecipitated solubilized alpha 2a receptor-Gi alpha complexes, indicating that all three Gi alpha subtypes couple with the alpha 2a receptor. Antiserum 9072, which is directed against the C-terminal region of G(o)alpha, immunoprecipitated solubilized alpha 2a receptor-G alpha complexes indicating that these receptors are also coupled to G(o)alpha. Antiserum 8132, which is directed against G beta 36, immunoprecipitated solubilized alpha 2a receptors while the G beta 35 antiserum 8129, did not, indicating that alpha 2a receptors selectively associate with G beta 36. The binding of the partial agonist p-aminoclonidine to the solubilized alpha 2a receptor alters the association of the receptor with G proteins. Following p-aminoclonidine binding to the solubilized alpha 2a receptor, the ability of the C-terminal directed G alpha antisera 8730 and 9072 to coimmunoprecipitate the alpha 2a receptor-G alpha complex was greatly reduced. The effect of p-aminoclonidine was concentration dependent, mimicked by the full agonist UK 14304 and blocked by the alpha 2 receptor antagonist yohimbine. In contrast, antisera directed against internal regions of Gi alpha and G(o)alpha, immunoprecipitated the agonist-bound and agonist-free alpha 2a receptor equally well. These findings indicate that following the binding of agonists to the alpha 2a receptor, Gi alpha and G(o)alpha remain physically associated with the receptor but either the conformation of G alpha linked to the receptor or the conformation of the receptor itself is modified such that the epitope for the C-terminal directed anti-Gi alpha and anti-G(o)alpha antisera are not accessible. These agonist-induced conformational changes in the alpha 2a receptor-G alpha complex may be important for the activation of the G protein and the stimulation of the alpha 2a receptor signal transduction pathway.     

A G protein gamma subunit peptide stabilizes a novel muscarinic receptor state

A prenylated peptide specific to the C terminal tail of a G protein gamma subunit type, gamma5, inhibits activation of a G protein by the M2 muscarinic receptor. The gamma5 peptide was tested for direct effects on the M2 receptor's properties. The wild type gamma5 peptide reduced the affinity of M2 for the agonist, carbachol, more than 5-fold in an antagonist displacement assay. The peptide was inactive when its amino acid sequence was scrambled or when it was unprenylated. Although the wild type peptide reduced the affinity of M2 for the antagonist QNB, it had no effect on the antagonists NMS or atropine. These results suggest that in the presence of the peptide the M2 receptor adopts a novel conformational state that affects the ligand binding surface. The results also suggest that the G protein gamma5 subunit tail interacts with a receptor.     

Functional domains of the 67-kDa laminin receptor precursor

We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin.     

Interaction of calmodulin with the serotonin 5-hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C

The 5-hydroxytryptamine2A (5-HT2A) receptor is a G(q/11)-coupled serotonin receptor that activates phospholipase C and increases diacylglycerol formation. In this report, we demonstrated that calmodulin (CaM) co-immunoprecipitates with the 5-HT2A receptor in NIH-3T3 fibroblasts in an agonist-dependent manner and that the receptor contains two putative CaM binding regions. The putative CaM binding regions of the 5-HT2A receptor are localized to the second intracellular loop and carboxyl terminus. In an in vitro binding assay peptides encompassing the putative second intracellular loop (i2) and carboxyl-terminal (ct) CaM binding regions bound CaM in a Ca2+-dependent manner. The i2 peptide bound with apparent higher affinity and shifted the mobility of CaM in a nondenaturing gel shift assay. Fluorescence emission spectral analyses of dansyl-CaM showed apparent K(D) values of 65 +/- 30 nM for the i2 peptide and 168 +/- 38 nM for the ct peptide. The ct CaM-binding domain overlaps with a putative protein kinase C (PKC) site, which was readily phosphorylated by PKC in vitro. CaM binding and phosphorylation of the ct peptide were found to be antagonistic, suggesting a putative role for CaM in the regulation of 5-HT2A receptor phosphorylation and desensitization. Finally, we showed that CaM decreases 5-HT2A receptor-mediated [35S]GTPgammaS binding to NIH-3T3 cell membranes, supporting a possible role for CaM in regulating receptor-G protein coupling. These data indicate that the serotonin 5-HT2A receptor contains two high affinity CaM-binding domains that may play important roles in signaling and function.     

Single amino acid of g(alpha16) (Ala(228)) is responsible for the ability of chemoattractant C5a receptor to induce G(alpha16)-mediated inositol phosphate release

Our previous study suggested that the region encompassing residues 220-240 on G(alpha16) is important in coupling with C5a receptor (Lee et al. (1995) Mol. Pharmacol. 47, 218-223). When aligned sequences are compared in the residue 220-240 segment of G(alpha16), there is a block of eight amino acids extending from residue 227 to residue 234 (227-Ile-Ala-Leu-Ile-Tyr-Leu-Ala-Ser-234) in G(alpha16) that is replaced by a heterologous block extending from amino acid residue 224 to residue 231 (224-Thr-Ser-Ile-Met-Phe-Leu-Val-Ala-231) in G(alpha11). In order to identify the specific amino acid residue necessary for coupling to C5a receptor within the extension of eight amino acids in G(alpha16), a series of chimeric G(alpha11)/G(alpha16) cDNA constructs and mutant G(alpha16) cDNAs were expressed. Then the ability of chimeras and mutant proteins to mediate C5a-induced release of inositol phosphate in transfected Cos-7 cells was tested. The results show that single amino acid Ala(228) is responsible for conferring about 40-50% of the activity of G(alpha16) induced by C5a receptor stimulation.     

Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.     

Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors.

Somatostatin (SRIF) induces its biological effects by interacting with membrane-bound receptors that are linked to cellular effector systems via G proteins. We have studied SRIF receptor-G protein associations by solubilizing the SRIF receptor from rat brain and AtT-20 cells and immunoprecipitating the receptor-G protein complex with peptide-directed antisera against the different subunits of the G protein heterotrimer. Antiserum 8730, which selectively interacts with all Gi alpha subtypes, maximally and specifically immunoprecipitated SRIF receptor-Gi alpha complexes. To identify the subtypes of Gi alpha that are coupled to SRIF receptors, the subtype-selective antisera 3646, 1521, and 1518, which specifically interact with Gi alpha 1, Gi alpha 2, and Gi alpha 3, respectively, were used to immunoprecipitate SRIF receptor-Gi alpha complexes. Antiserum 3646 immunoprecipitated SRIF receptor-Gi alpha 1 complexes from both brain and AtT-20 cells. Antiserum 1521 immunoprecipitated Gi alpha 2 from both brain and AtT-20 cells but did not immunoprecipitate SRIF receptors from these tissues. Antiserum 1518 immunoprecipitated AtT-20 cell SRIF receptors but uncoupled brain SRIF receptor-G protein complexes. This result was confirmed with another peptide-selective antiserum, SQ, directed against Gi alpha 3. The findings from these studies indicate that Gi alpha 1 and Gi alpha 3 are coupled to SRIF receptors, whereas Gi alpha 2 is not. Even though brain and AtT-20 cell SRIF receptors were both coupled to Gi alpha, the receptors from these tissues differed in their coupling to Go alpha. Antiserum 2353, which is directed against Go alpha, immunoprecipitated SRIF receptors from AtT-20 cells, but did not immunoprecipitate or uncouple SRIF receptor-G protein complexes from rat brain. To determine the beta subunits associated with the SRIF receptor, antisera directed against G beta 36 and G beta 35 were used to immunoprecipitate SRIF receptor-G protein complexes from brain. Peptide-directed antiserum against G beta 36 selectively immunoprecipitated solubilized brain SRIF receptors. However, antiserum directed against the G beta 35 subunit did not immunoprecipitate brain SRIF receptors, suggesting that brain SRIF receptors may preferentially associate with G beta 36. In addition to coimmunoprecipitating with Gi alpha and G beta, brain SRIF receptors coimmunoprecipitated the G protein gamma subunits, G gamma 2 and G gamma 3. These results provide the first evidence that SRIF receptors are coupled to different subunits of G proteins and suggest that selectivity exists in the association of different G protein subunits with the SRIF receptor.     

Brain somatostatin receptor-G protein interaction. G alpha C-terminal antibodies demonstrate coupling of the soluble receptor with Gi(1-3) but not with Go.

Somatostatin (SST) receptors activate potassium channels, stimulate protein phosphatases, inhibit adenylate cyclase and close calcium channels. These multiple effects are controlled by guanine nucleotide binding (G) proteins of the pertussis toxin-sensitive Gi and Go types. In the present study we have identified the G proteins coupling with brain SST receptors. To this end, brain SST receptors were solubilized in G-protein coupled form. Binding of the SST analogue MK 678 to the solubilized receptor was completely inhibited by guanosine 5'-O-thiotriphosphate (IC50 = 100 nM), reflecting decreased receptor affinity for agonist following uncoupling of the receptor and G protein(s). Antibodies raised against specific COOH-terminal peptides of the G proteins Gi(1-3), Go, and Gz were used to probe for SST receptor-G protein coupling in this system. Antibodies binding to the COOH-terminal regions of Gi1 and Gi2 (antibody AS) and Gi3 (antibody EC) inhibited binding of 125I-MK 678 (75 pM) by 57 +/- 4% and 48 +/- 5%, respectively. The effects of these antibodies were concentration-dependent and additive, such that in combination AS and EC completely inhibited binding. Antibodies binding to the COOH-terminal region of Go (GO) and Gz (QN) did not affect binding of 125I-MK 678, indicating that neither Go nor Gz are associated with the brain SST receptor. Prelabeling of the receptor with 125I-MK 678 prior to addition of antibody induced the formation of a "locked conformation" of the agonist-bound receptor-G protein complex which was insensitive to antibody. In conclusion, Gi1 and/or Gi2 and Gi3 are coupled in approximately equal proportions to the brain 125I-MK 678-binding SST receptor, accounting for all of the G protein coupling of this receptor.     

Tertiary structure of human complement component C5a in solution from nuclear magnetic resonance data

The tertiary structure for the region 1-63 of the 74 amino acid human complement protein C5a in solution was calculated from a large number of distance constraints derived from nuclear Overhauser effects with an angular distance geometry algorithm. The protein consists of four helices juxtaposed in an approximately antiparallel topology connected by peptide loops located at the surface of the molecule. The structures obtained for the helices are compatible with alpha-helical hydrogen-bonding patterns, which provides an explanation for the observed slow solvent exchange kinetics of the amide protons in these peptide regions. In contrast to the peptide region 1-63, no defined structure could be assigned to the C-terminal region 64-74, which increasingly acquires dynamic random coil characteristics as the end of the peptide chain is approached. An average root-mean-square deviation of 1.6 A was obtained for the alpha-carbons of the first 63 residues in the calculated ensemble of C5a structures, while the alpha-helices were determined with an average root-mean-square deviation of 0.8 A for the alpha-carbons. A comparison between the solution structure of C5a and the crystal structure of the functionally related C3a protein, as well as inferences for the interaction of C5a with its receptor on polymorphonuclear leukocytes, is discussed.     

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.     

Localization of the amino-acid exchange in protein S5 from an Escherichia coli mutant resistant to spectinomycin

Summary Ribosomal proteins S5 were isolated from E. coli B wild type and from a spectinomycin resistant mutant derived from it. After tryptic digestion the peptides were isolated and their amino acid compositions compared. An amino acid replacement, namely arginine by leucine, was found at the C-terminus of peptide T8. This result, together with our previous studies, shows that in spectinomycin resistant mutants the amino acid replacements are clustered within a very narrow region of protein S5.     

Theoretical study of the electrostatically driven step of receptor-G protein recognition

This study proposes a theoretical model describing the electrostatically driven step of the alpha 1 b-adrenergic receptor (AR)-G protein recognition. The comparative analysis of the structural-dynamics features of functionally different receptor forms, i.e., the wild type (ground state) and its constitutively active mutants D142A and A293E, was instrumental to gain insight on the receptor-G protein electrostatic and steric complementarity. Rigid body docking simulations between the different forms of the alpha 1 b-AR and the heterotrimeric G alpha q, G alpha s, G alpha i1, and G alpha t suggest that the cytosolic crevice shared by the active receptor and including the second and the third intracellular loops as well as the cytosolic extension of helices 5 and 6, represents the receptor surface with docking complementarity with the G protein. On the other hand, the G protein solvent-exposed portions that recognize the intracellular loops of the activated receptors are the N-terminal portion of alpha 3, alpha G, the alpha G/alpha 4 loop, alpha 4, the alpha 4/beta 6 loop, alpha 5, and the C-terminus. Docking simulations suggest that the two constitutively active mutants D142A and A293E recognize different G proteins with similar selectivity orders, i.e., G alpha q approximately equal to G alpha s > G alpha i > G alpha t. The theoretical models herein proposed might provide useful suggestions for new experiments aiming at exploring the receptor-G protein interface.