The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Origins of the specificity of tissue-type plasminogen activator.

The role of subsite interactions in defining the stringent substrate specificity of tissue-type plasminogen activator (t-PA) has been examined by using an fd phage library that displayed random hexapeptide sequences and contained 2 x 10(8) independent recombinants. Forty-four individual hexapeptides were isolated and identified as improved substrates for t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide that contained the primary sequence of the actual cleavage site in plasminogen. These results suggest that small peptides can mimic determinants that mediate specific proteolysis, emphasize the importance of subsite interactions in determining protease specificity, and have important implications for the evolution of protease cascades.

Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximately 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximately 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. Sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.

Tissue specificity of a glucocorticoid-dependent enhancer in transgenic mice.

The glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase were associated with the regulatory sequences of a cellular gene expressed ubiquitously--that coding for the largest subunit of RNA polymerase II. In transient expression assays, glucocorticoid responsiveness of the hybrid regulatory regions depends on the spatial relationship and number of regulatory elements. Two parameters affect the ratio of induction by glucocorticoids: the basal level of the hybrid promoter that is affected by the RNA polymerase II regulatory sequences and the glucocorticoid-induced level that depends on the distance between the GRUs and the TATA box. A fully active glucocorticoid-responsive hybrid gene was used to generate transgenic mice. Results show that a composite regulatory pattern is obtained: ubiquitous basal expression characteristic of the RNA polymerase II gene and liver-specific glucocorticoid activation characteristic of the tyrosine aminotransferase GRUs. This result demonstrates that the activity of the tyrosine aminotransferase GRUs is cell-type-specific not only in cultured cells but also in the whole animal.

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.

Modification of Ser59 in the unique N-terminal region of tyrosine kinase p56lck regulates specificity of its Src homology 2 domain.

During T-cell activation, Ser59 in the unique N-terminal region of p56lck is phosphorylated. Mutation of Ser59 to Glu59 mimics Ser59 phosphorylation, and upon CD4 crosslinking, this mutant p56lck induces tyrosine phosphorylation of intracellular proteins distinct from those induced by wild-type p56lck. Mutant and wild-type p56lck have similar affinities for CD4 and similar kinase activities. In glutathione S-transferase fusion proteins, the p56lck Src homology 2 (SH2) domain with the SH3 domain and the unique N-terminal region (including Ser59) has a different binding specificity for phosphotyrosyl proteins than the SH2 domain alone. Either deletion of the unique N-terminal region or mutation of Ser59 to Glu59 in the fusion protein reverts the phosphotyrosyl protein binding specificity back to that of the SH2 domain alone. These results suggest that phosphorylation of Ser59 regulates the function of p56lck by controlling binding specificity of its SH2 domain.

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Strand specificity in the transcriptional targeting of recombination at immunoglobulin switch sequences.

B-lymphocyte-specific class switch recombination is known to occur between pairs of 2- to 10-kb switch regions located immediately upstream of the immunoglobulin constant heavy-chain genes. Others have shown that the recombination is temporally correlated with the induction of transcription at the targeted switch regions. To determine whether this temporal correlation is due to a mechanistic linkage, we have developed an extrachromosomal recombination assay that closely recapitulates DNA deletional class switch recombination. In this assay, the rate of recombination is measured between 24 and 48 hr posttransfection. We find that recombinants are generated in a switch sequence-dependent manner. Recombination occurs with a predominance within B-cell lines representative of the mature B-cell stage and within a subset of pre-B-cell lines. Transcription stimulates the switch sequence-dependent recombination. Importantly, transcription activates recombination only when directed in the physiologic orientation but has no effect when directed in the nonphysiologic orientation.

Correlation of peptide specificity and IgG subclass with pathogenic and nonpathogenic autoantibodies in pemphigus vulgaris: a model for autoimmunity.

Pemphigus vulgaris (PV) is a rare, potentially fatal, autoimmune disease that affects the skin and mucous membranes. The PV antigen (PVA) has been characterized as desmoglein 3. PV patients carry HLA-DR4- or HLA-DR6-bearing extended haplotypes. We recently demonstrated that patients with active disease have high titers of PV autoantibodies of the IgG1 and IgG4 subclasses. Patients in remission, healthy unaffected relatives, and some MHC-matched normal individuals have low levels of PV autoantibodies, which are IgG1 only. Furthermore, intraperitoneal injection of IgG from patients with active disease caused clinical disease in mice, but IgG from patients in remission, healthy relatives, or MHC-matched normal individuals did not. We prepared 12 peptides of 30 amino acids each (peptides Bos 1-12) spanning the extracellular domain of PVA. Patients with active disease recognize peptides Bos 1 and Bos 6 with high titers of IgG1 and IgG4 autoantibodies. Patients in remission have IgG1 autoantibodies to peptide Bos 1 only, in statistically significantly lower titers (P < 0.01). They no longer have IgG4 subclass autoantibodies to peptide Bos 6. Healthy relatives and normal unrelated individuals have low levels of only IgG1 autoantibodies that recognize only Bos 1. In vitro studies indicate that Bos 6-specific IgG and, to a lesser extent, Bos 1-specific IgG can cause acantholysis. Our data suggest that Bos 6-specific IgG4 is probably the main acantholytic autoantibody, while Bos 1-specific IgG4 may act as a facilitator or enhancer of the process. In this study we illustrate some of the paradigms that demonstrate the interactions between the MHC, subclass of autoantibodies, and peptide specificities of the autoantibodies in the autoimmune process. Thus, PV provides an important model to study the pathogenesis of autoimmunity.

Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.

Disease specificity of kinase domains: the src-encoded catalytic domain converts erbB into a sarcoma oncogene.

src and erbB are two tyrosine kinase-encoding oncogenes carried by retroviruses, which have distinct disease specificities. The former induces predominantly sarcomas, and the latter, leukemias. Src and ErbB have similar catalytic domains but have very different regulatory domains. A wealth of information exists concerning how different regulatory domains [Src homology 2 (SH2) and SH3 domains and autophosphorylation sites] control substrate and disease specificities. Whether the catalytic domain helps determine these specificities remains to be explored. Here we show that the Src catalytic domain is enzymatically active when substituted into the ErbB backbone and interacts with the ErbB regulatory domain. This ErbB/Src chimera displays autophosphorylation and substrate phosphorylation patterns different from those of both Src and ErbB. Neither SH2 and SH3 nor autophosphorylation sites are required for the Src catalytic domain to exert its fibroblast transforming ability. Most significantly, the catalytic domain can convert erbB from a leukemogenic oncogene into a sarcomagenic oncogene, suggesting that the leukemogenic determinants in part reside within the ErbB catalytic domain.

Heterogeneity in molecular recognition by restriction endonucleases: osmotic and hydrostatic pressure effects on BamHI, Pvu II, and EcoRV specificity.

The cleavage specificity of the Pvu II and BamHI restriction endonucleases is found to be dramatically reduced at elevated osmotic pressure. Relaxation in specificity of these otherwise highly accurate and specific enzymes, previously termed "star activity," is uniquely correlated with osmotic pressure between 0 and 100 atmospheres. No other colligative solvent property exhibits a uniform correlation with star activity for all of the compounds tested. Application of hydrostatic pressure counteracts the effects of osmotic pressure and restores the natural selectivity of the enzymes for their canonical recognition sequences. These results indicate that water solvation plays an important role in the site-specific recognition of DNA by many restriction enzymes. Osmotic pressure did not induce an analogous effect on the specificity of the EcoRV endonuclease, implying that selective hydration effects do not participate in DNA recognition in this system. Hydrostatic pressure was found to have little effect on the star activity induced by changes in ionic strength, pH, or divalent cation, suggesting that distinct mechanisms may exist for these observed alterations in specificity. Recent evidence has indicated that BamHI and EcoRI share similar structural motifs, while Pvu II and EcoRV belong to a different structural family. Evidently, the use of hydration water to assist in site-specific recognition is a motif neither limited to nor defined by structural families.

Straight but not Narrow; Within-Gender Variation in the Gender-Specificity of Women's Sexual Response

Gender differences in the specificity of sexual response have been a primary focus in sexual psychophysiology research, however, within-gender variability suggests sexual orientation moderates category-specific responding among women; only heterosexual women show gender-nonspecific genital responses to sexual stimuli depicting men and women. But heterosexually-identified or “straight” women are heterogeneous in their sexual attractions and include women who are exclusively androphilic (sexually attracted to men) and women who are predominantly androphilic with concurrent gynephilia (sexually attracted to women). It is therefore unclear if gender-nonspecific responding is found in both exclusively and predominantly androphilic women. The current studies investigated within-gender variability in the gender-specificity of women’s sexual response. Two samples of women reporting concurrent andro/gynephilia viewed (Study 1, n = 29) or listened (Study 2, n = 30) to erotic stimuli varying by gender of sexual partner depicted while their genital and subjective sexual responses were assessed. Data were combined with larger datasets of predominantly gyne- and androphilic women (total N = 78 for both studies). In both studies, women reporting any degree of gynephilia, including those who self-identified as heterosexual, showed significantly greater genital response to female stimuli, similar to predominantly gynephilic women; gender-nonspecific genital response was observed for exclusively androphilic women only. Subjective sexual arousal patterns were more variable with respect to sexual attractions, likely reflecting stimulus intensity effects. Heterosexually-identified women are therefore not a homogenous group with respect to sexual responses to gender cues. Implications for within-gender variation in women’s sexual orientation and sexual responses are discussed.

The specificity of emotion inferences as a function of emotional contextual support

Research on emotion inferences has shown that readers include a representation of the main character's emotional state in their mental representations of the text. We examined the specificity of emotion representations as a function of the emotion content of short narratives, in terms of the quantity and quality of emotion components included in the narratives, based on the GRID instrument (Fontaine et al., 2013). In a self-paced reading task, target sentences that only moderately matched the emotional context were read faster than target sentences that strongly matched the emotional context of the narratives. In a “makes sense” judgment task, we showed that this result was not driven by a mapping difficulty and, in a memory task, we provided some evidence that these effects reflected integration processes. We suggest that readers can integrate specific emotions into their mental representations, but only if provided with the appropriate emotional contextual support.