Cloning and characterization of the human galanin GALR2 receptor

We present the molecular cloning and characterization of the human galanin receptor, hGALR2. hGALR2 shares 85%, 39%, and 57% amino acid identities to rGALR2, hGALR1, and hGALR3, respectively. hGALR2, along with rGALR2, can be distinguished from the other cloned galanin receptors by a tolerance for both N-terminal extension and C-terminal deletion of galanin, as well as by a primary signaling mechanism involving phosphatidyl inositol hydrolysis and calcium mobilization. By RT-PCR, GALR2 mRNA was abundant in human hippocampus, hypothalamus, heart, kidney, liver, and small intestine. A weak GALR2 mRNA signal was detected in human retina, and no signal was detected in cerebral cortex, lung, spleen, stomach, or pituitary.     

Analysis of human ITPase nucleobase specificity by site-directed mutagenesis

Inosine triphosphate (ITP) pyrophosphohydrolase, or ITPase, is an intracellular enzyme that is responsible for the hydrolysis of the acidic anhydride bond between the alpha and beta phosphates in ITP, and other noncanonical nucleoside triphosphates, producing the corresponding nucleoside monophosphate and pyrophosphate. This activity protects the cell by preventing noncanonical nucleoside triphosphates from accumulating in (deoxy) nucleoside triphosphate ((d)NTP) pools and/or being integrated into nucleic acids. This enzyme is encoded by the ITPA gene in mammals. It has been reported that Itpa homozygous-null knock-out mice die before weaning and have gross cardiac abnormalities. Additionally, certain variations in the human ITPA gene have been linked to adverse reactions to the immunosuppressive prodrugs azathioprine and 6-mercaptopurine and protection against ribavirin-induced hemolytic anemia. These drugs are bioactivated to form noncanonical nucleoside triphosphates. Human ITPase enzymes engineered to modulate nucleobase specificity may be valuable tools for studying the role of ITPase in heart development and drug metabolism or developing gain-of-function mutants or inhibitory molecules. Based on x-ray crystallography and amino acid sequence data, a panel of putative human ITPase nucleobase specificity mutants has been generated. We targeted eight highly conserved amino acid positions within the ITPase sequence that correspond to amino acids predicted to directly interact with the nucleobase or help organize the nucleobase binding pocket. The ability of the mutants to protect against exogenous and endogenous noncanonical purines was tested with two Escherichia coli complementation assays. Nucleobase specificity of the mutants was investigated with an in vitro biochemical assay using ITP, GTP and ATP as substrates. This methodology allowed us to identify gain-of-function mutants and categorize the eight amino acid positions according to their ability to protect against noncanonical purines as follows: Glu-22, Trp-151 and Arg-178, essential for protection; Phe-149, Asp-152, Lys-172 and Ser-176, intermediate protection; His-177, dispensable for protection against noncanonical purines.     

Probing the determinants of coenzyme specificity in ferredoxin-NADP+ reductase by site-directed mutagenesis

On the basis of sequence and three-dimensional structure comparison between Anabaena PCC7119 ferredoxin-NADP(+) reductase (FNR) and other reductases from its structurally related family that bind either NADP(+)/H or NAD(+)/H, a set of amino acid residues that might determine the FNR coenzyme specificity can be assigned. These residues include Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235. Systematic replacement of these amino acids was done to identify which of them are the main determinants of coenzyme specificity. Our data indicate that all of the residues interacting with the 2'-phosphate of NADP(+)/H in Anabaena FNR are not involved to the same extent in determining coenzyme specificity and affinity. Thus, it is found that Ser-223 and Tyr-235 are important for determining NADP(+)/H specificity and orientation with respect to the protein, whereas Arg-224 and Arg-233 provide only secondary interactions in Anabaena FNR. The analysis of the T155G FNR form also indicates that the determinants of coenzyme specificity are not only situated in the 2'-phosphate NADP(+)/H interacting region but that other regions of the protein must be involved. These regions, although not interacting directly with the coenzyme, must produce specific structural arrangements of the backbone chain that determine coenzyme specificity. The loop formed by residues 261-268 in Anabaena FNR must be one of these regions.     

Molecular modelling and site-directed mutagenesis on a bovine anti-testosterone monoclonal antibody.

A three-dimensional (3D) molecular model of the antigen-combining site of a bovine anti-testosterone monoclonal antibody has been constructed. In the model, the CDRs, and a single heavy chain framework region residue (Trp47), associate to form a hydrophobic cavity large enough to accommodate a single molecule of testosterone. Tyr97 of CDR-H3 lies at the bottom of the cavity with its hydroxyl group exposed to solvent. Using the model and data from binding studies, we predicted that the cavity forms the antibody's paratope and on binding testosterone a hydrogen bond is formed between Tyr97 of CDR-H3 and the hydroxyl group on the D-ring of testosterone. This prediction has subsequently been tested by site-directed mutagenesis. An antibody with phenylalanine in place of tyrosine at position 97 in CDR-H3 has its affinity reduced by approximately 800 fold. The reduction in binding energy associated with the reduced affinity has been calculated to be 3.9 kcal/mol which is within the range (0.5-4.0 kcal/mol) expected for the loss of a single hydrogen bond. The model has been used to suggest ways of increasing the antibody's affinity for testosterone.     

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis

Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptostreptococcus asaccharolyticus has a strong preference for NADH over NADPH as a coenzyme, over 1000-fold in terms of kcat/Km values. Sequence alignments across the wider family of NAD(P)-dependent dehydrogenases might suggest that this preference is mainly due to a negatively charged glutamate at position 243 (E243) in the adenine ribose-binding pocket. We have examined the possibility of altering coenzyme specificity of the Peptostreptococcus enzyme, and, more specifically, the role of residue 243 and neighbouring residues in coenzyme binding, by introducing a range of point mutations. Glutamate dehydrogenases are unusual among dehydrogenases in that NADPH-specific forms usually have aspartate at this position. However, replacement of E243 with aspartate led to only a nine-fold relaxation of the strong discrimination against NADPH. By contrast, replacement with a more positively charged lysine or arginine, as found in NADPH-dependent members of other dehydrogenase families, allows a more than 1000-fold shift toward NADPH, resulting in enzymes equally efficient with NADH or NADPH. Smaller shifts in the same direction were also observed in enzymes where a neighboring tryptophan, W244, was replaced by a smaller alanine (approximately six-fold) or Asp245 was changed to lysine (32-fold). Coenzyme binding studies confirm that the mutations result in the expected major changes in relative affinities for NADH and NADPH, and pH studies indicate that improved affinity for the extra phosphate of NADPH is the predominant reason for the increased catalytic efficiency with this coenzyme. The marked difference between the results of replacing E243 with aspartate and with positive residues implies that the mode of NADPH binding in naturally occurring NADPH-dependent glutamate dehydrogenases differs from that adopted in E243K or E243D and in other dehydrogenases.     

Reactive-site specificity of human kallistatin toward tissue kallikrein probed by site-directed mutagenesis

Kallistatin is a serine proteinase inhibitor that forms complexes with tissue kallikrein and inhibits its activity. In this study, we compared the inhibitory activity of recombinant human kallistatin and two mutants, Phe388Arg (P1) and Phe387Gly (P2), toward human tissue kallikrein. Recombinant kallistatins were expressed in Escherichia coli and purified to apparent homogeneity using metal-affinity and heparin-affinity chromatography. The complexes formed between recombinant kallistatins and tissue kallikrein were stable for at least 150 h. Wild-type kallistatin as well as both Phe388Arg and Phe387Gly mutants act as inhibitors and substrates to tissue kallikrein as analyzed by complex formation. Kinetic analyses showed that the inhibitory activity of Phe388Arg variant toward tissue kallikrein is two-fold higher than that of wild type (P1Phe), whereas Phe387Gly had only 7% of the inhibitory activity toward tissue kallikrein as compared to wild type. The Phe388Arg variant but not wild type inhibited plasma kallikrein's activity. These results indicate that P1Arg variant exhibits more potent inhibitory activity toward tissue kallikrein while wild type (P1Phe) is a more selective inhibitor of tissue kallikrein. The P2 phenylalanine is essential for retaining the hydrophobic environment for the interaction of kallistatin and kallikrein.     

Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis

Xylose isomerases (XIs) from Thermoanaerobacterium thermosulfurigenes (TTXI) and Thermotoga neapolitana (TNXI) are 70.4% identical in their amino acid sequences and have a nearly superimposable crystal structure. Nonetheless, TNXI is much more thermostable than TTXI. Except for a few additional prolines and fewer Asn and Gln residues in TNXI, no other obvious differences in the enzyme structures can explain the differences in their stabilities. TNXI has two additional prolines in the Phe59 loop (Pro58 and Pro62). Mutations Gln58Pro, Ala62Pro and Gln58Pro/Ala62Pro in TTXI and their reverse counterpart mutations in TNXI were constructed by site-directed mutagenesis. Surprisingly, only the Gln58Pro mutation stabilized TTXI. The Ala62Pro and Gln58Pro/Ala62Pro mutations both dramatically destabilized TTXI. Analysis of the three-dimensional (3D) structures of TTXI and its Ala62Pro mutant derivative showed a close van der Waal's contact between Pro62-C(delta) and atom Lys61-C(beta) (2.92 A) thus destabilizing TTXI. All the reverse counterpart mutations destabilized TNXI thus confirming that these two prolines play important roles in TNXI's thermostability. TTXI's active site has been previously engineered to improve its catalytic efficiency toward glucose and increase its thermostability. The same mutations were introduced into TNXI, and similar trends were observed, but to different extents. Val185Thr mutation in TNXI is the most efficient mutant derivative with a 3.1-fold increase in its catalytic efficiency toward glucose. With a maximal activity at 97 degrees C of 45.4 U/mg on glucose, this TNXI mutant derivative is the most active type II XI ever reported. This 'true' glucose isomerase engineered from a native xylose isomerase has now comparable kinetic properties on glucose and xylose.     

Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions

To define the specific role of the galanin receptors when mediating the effect of galanin, effective tools for distinct activation and inhibition of the different receptor subtypes are required. Several of the physiological effects modulated by galanin are implicated to be mediated via the GalR2 subtype and have been distinguished from GalR1 effects by utilizing the Gal(2–11) peptide, recognizing only GalR2 and GalR3. In this study, we have performed a mutagenesis approach on the GalR2 subtype and present, for the first time, a molecular characterization of the interactions responsible for ligand binding and receptor activation at this receptor subtype. Our results identify four residues, His²⁵² and His²⁵³ located in transmembrane domain 6 and Phe²⁶⁴ and Tyr²⁷¹ in the extracellular loop 3, to be of great significance. We show evidence for the N-terminal tail of GalR2 to participate in ligand binding and that selective binding of Gal(2–11) includes interaction with the Ile²⁵⁶ residue, located at the very top of TM 6. In conclusion, we present a mutagenesis study on GalR2 and confer interactions responsible for ligand binding and receptor activation as well as selective recognition of the Gal(2–11) peptide at this receptor subtype. The presented observations could be of major importance for the design and development of new and improved peptide and non-peptide ligands, selectively activating the GalR2 subtype.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2alpha), which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of CK2alpha and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in CK2 alpha subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known CK2alpha. The mutant deltaN118, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased k(cat), possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208.     

Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis

The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.     

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of k_cat values of the wild-type enzyme for the preferred substrates, -leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids.     

Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: site-directed mutagenesis and kinetic studies.

Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics. This reaction is catalyzed by a family of enzymes named sulfotransferases. The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities. SULT1A1 favors simple phenolic substrates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates such as dopamine. In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3. First, the mutation A146E in SULT1A1 yielded a SULT1A3-like protein with respect to the Michaelis constant for simple phenols. The mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respect to the Michaelis constant for both simple phenols and monoamine compounds. When comparing the specificity of SULT1A3 toward tyramine with that for p-ethylphenol (which differs from tyramine in having no amine group on the carbon side chain), we saw a 200-fold preference for tyramine. The kinetic data obtained with the E146A mutant of SULT1A3 for these two substrates clearly showed that this protein preferred substrates without an amine group attached. Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resulted in a 360-fold decrease in the specificity constant for dopamine. The results provide strong evidence that residue 146 is crucial in determining the substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates.     

Investigating the structure of human RNase H1 by site-directed mutagenesis

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.     

Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis

Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.     

Phenotypic analysis of mice deficient in the type 2 galanin receptor (GALR2)

Galanin is a neuropeptide implicated in the regulation of feeding, reproduction, cognition, nociception, and seizure susceptibility. There are three known galanin receptor (GALR) subtypes (GALR1, GALR2, and GALR3), which bind to galanin with different affinities and have their own unique distributions, signaling mechanisms, and putative functions in the brain and peripheral nervous system. To gain further insight into the possible physiological significance of GALR2, we created mutant mice that were deficient in GALR2 and compared their phenotype to that of wild-type (WT) littermate or age-matched controls, with respect to basic motor and sensory function, feeding behavior, reproduction, mood, learning and memory, and seizure susceptibility. Phenotypic analysis revealed that animals bearing a deletion of GALR2 did not differ significantly from their WT controls in any of the measured variables. We conclude that either GALR2 plays no role in these physiological functions or through redundancy or compensation these mutant animals can adapt to the congenital absence of GALR2. It is also conceivable that GALR2 plays only a subtle role in some of these functions and that the impact of its loss could not be detected by the analytical procedures used here.     

Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase: Analysis by site-directed mutagenesis

A comparison of laccase sequences highlighted the presence of a C-terminal extension of sixteen amino acids in POXA1b laccase – that represents the most thermostable isoenzyme among Pleurotus ostreatus laccases and exhibits a notable stability at alkaline pH (t_1/2 at pH 10 = 30 days) – whereas this tail is missing in the other analysed laccases from basidiomycetes. Site-directed mutagenesis experiments allowed us to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. The truncated mutants lose the high stability at pH 10, while they show an increased stability at pH 5. The effect of substituting the residue Asp205 of POXA1b with an arginine was also analysed in the mutant POXA1bD205R. Following the mutation POXA1bD205R, a remarkable worsening of catalytic properties along with a decrease of substrate affinity and of enzyme stability were found. It was demonstrated that introducing Arg205 mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein.     

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis.

Site-specific mutagenesis was employed to study structure-function relationships at the substrate binding site of rat tissue kallikrein. Four kallikrein mutants, the Pro219 deletion (P219del), the 34-38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34-38)IN], the Trp215----Gly exchange (W215G) and the double mutant with Tyr99----His and Trp215----Gly exchange (Y99H:W215G) were created by site-directed mutagenesis to probe their function in substrate binding. The mutant proteins were expressed in Escherichia coli at high levels and analyzed by Western blot. These mutant enzymes were purified to apparent homogeneity. Each migrated as a single band on SDS-PAGE, with slightly lower molecular mass (36 kDa) than that of the native enzyme, (38 kDa) because of their lack of glycosylation. The recombinant kallikreins are immunologically identical to the native enzyme, displaying parallelism with the native enzyme in a direct radioimmunoassay for rat tissue kallikrein. Kinetic analyses of Km and kcat using fluorogenic peptide substrates support the hypothesis that the Tyr99-Trp215 interaction is a major determinant for hydrophobic P2 specificity. The results suggest an important role for the 34-38 loop in hydrophobic P3 affinity and further show that Pro219 is essential to substrate binding and efficient catalysis of tissue kallikrein.     

Intracerebroventricular administration of galanin or galanin receptor subtype 1 agonist M617 induces c-Fos activation in central amygdala and dorsomedial hypothalamus

The neuropeptide galanin and galanin receptors are widespread throughout cortical, limbic and midbrain areas implicated in reward, learning/memory, pain, drinking and feeding. While many studies have shown that galanin produces a variety of presynaptic and post-synaptic responses, work studying the effects of galanin on neural activation is limited. The present study examined patterns of c-Fos immunoreactivity resulting from intracerebroventricular administration of galanin versus saline injection in awake rats. An initial comprehensive qualitative survey was conducted to identify regions of high c-Fos expression followed up with quantitative analysis. Galanin induced a significant increase in c-Fos levels relative to saline-treated controls in dorsomedial hypothalamus and in the central nucleus of the amygdala. This pattern of activation was also produced by galanin receptor type 1 agonist M617. The present findings confirm that galanin upregulates c-Fos activation in hypothalamic nuclei, and supports roles for galanin in central amygdala-mediated regulation of stress-responses, food intake, and Pavlovian conditioning.     

Mapping of the functional determinants of the integrin beta 1 cytoplasmic domain by site-directed mutagenesis.

We describe here the expression of deletion mutants of the cytoplasmic domain of the avian integrin beta 1 subunit. These mutants, which contain termination codons at positions 767, 776, 791, and 800, were transfected into mouse 3T3 cells to determine which sequences were essential for localization of integrins into focal contact sites. In all cases, high-level expression of the truncated avian integrins was obtained. Heterodimers were formed between the exogenous truncated avian beta 1 subunits and endogenous mouse alpha subunits, and these heterodimers were efficiently exported to the cell surface. The longest truncated beta 1 subunit tested, which is only four amino acids shorter than the wild type, does localize to focal contacts. In contrast, beta 1 subunits with moderately long truncations of the cytoplasmic domain failed to localize to focal contacts, including one which contains the consensus sequence for tyrosine phosphorylation. Surprisingly, a mutant subunit in which the bulk of the cytoplasmic domain was missing (but the segment nearest the membrane including the dibasic residues (RR) remained) did localize weakly to focal contacts. These results implicate the peptide segment nearest to the transmembrane region in focal contact localization. In addition, mutant subunits that included this segment together with a larger portion of the cytoplasmic domain did not localize as well as the shorter form, suggesting that these cytoplasmic domain segments are defective, presumably because of abnormal folding.