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PubMed2027

Identification of Genome-Wide Chromatin Structural Aberration in Cancer by Hi-C Analysis.

Aberrant three-dimensional genome organization is a hallmark of cancer, often driving oncogene activation through mechanisms such as enhancer hijacking. High-throughput chromosome conformation capture (Hi-C) maps these interactions on a genome-wide scale. Unlike earlier dilution-based methods, in situ Hi-C performs proximity ligation within intact nuclei, minimizing random ligation noise and enabling fine-scale structure detection. This chapter describes an optimized in situ Hi-C protocol tailored for cancer cell lines using MboI digestion and biotin-mediated pull-down to generate high-complexity libraries. We further outline a computational workflow that extends beyond standard topological mapping of compartments and topologically associating domains to identify cancer-specific aberrations. Specifically, we focus on detecting chromosomal rearrangements (structural variants) and characterizing the distinct circular topology of extrachromosomal DNA. This integrated experimental and analytical framework provides the necessary tools to dissect the spatial dysregulation underlying tumor evolution.

Humans↗

PubMed1989

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain.

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.

Adenosine Triphosphate↗

PubMed1999

Identification of a cyclic nucleotide- and voltage-activated ion channel from insect antennae.

From an antennal cDNA library of Heliothis virescens a clone has been isolated encoding a polypeptide of 678 amino acids. Data base comparisons and primary structure analysis of the deduced protein sequence (HvCNG) indicated significant homology to cyclic nucleotide and voltage-activated ion channels including six putative membrane spanning domains, a putative cyclic nucleotide binding site, a pore region and a voltage-sensor motif. Heterologous expression of the cloned cDNA in Sf9 cells resulted in a polypeptide of the predicted molecular mass. Patch clamp analysis allowed to record the activity of the identified HvCNG channels; they were activated by cAMP but also by hyperpolarization. The channel displayed in potassium solution a conductance of 30 pS; the ion selectivity was calculated as PK/PNa approximately 3. Northern blot analysis revealed that the channel is highly expressed in the antennae; weaker signal were detected in heads and legs. In situ hybridization of tissue sections through the antennae showed a spatial distribution of reactive cells; they are located beneath sensillar hairs. Thus, a novel channel type has been identified which may play an important role in antennal cells.

Amino Acid Sequence↗

PubMed1996

Characterization of EZH1, a human homolog of Drosophila Enhancer of zeste near BRCA1.

Recent transcription mapping efforts within chromosome 17q21 have led to the identification of a human homolog of the Drosophila gene Enhancer of zeste, E(z). A member of the Polycomb group (Pc-G) of proteins, Drosophila E(z) acts as a negative regulator of the segment identity genes of the Antennapedia and Bithorax complexes. Here we report the full-length protein coding sequence of human EZH1 (Enhancer of zeste homolog 1) and compare the respective protein sequences in both species. EZH1 encodes a protein of 747 amino acids that displays 55% amino acid identity overall (70% similarity) with Drosophila E(z). The strongest homology was noted (79% identity, 89% similarity) within the carboxy-terminal 245 amino acids, including the SET domain, a region of E(z) also conserved in other Drosophila proteins with roles in development and/or chromatin structure. A large Cysrich region with a novel spatial pattern of cysteine residues was also conserved in both EZH1 and E(z). The strong sequence conservation suggest potential roles for EZH1 in human development as a transcriptional regulator and as a component of protein complexes that stably maintain heterochromatin. EZH1 is expressed as two major transcripts in all adult and fetal human tissues surveyed; comparison of cloned cDNAs suggests that alternative splicing may account for at least part of the transcript size difference. Analysis of one cDNA revealed an unusual splicing event involving EZH1 and a tandemly linked gene GPR2 and suggests a potential mechanism for modifying the EZH1 protein in the conserved C-terminal domain. The sequence and isolated cDNAs will provide useful reagents for determining the function of EZH1 and the importance of the evolutionarily conserved domains.

Adult↗

PubMed2004

The Speech, Spatial and Qualities of Hearing Scale (SSQ).

The Speech, Spatial and Qualities of Hearing Scale (SSQ) is designed to measure a range of hearing disabilities across several domains. Particular attention is given to hearing speech in a variety of competing contexts, and to the directional, distance and movement components of spatial hearing. In addition, the abilities both to segregate sounds and to attend to simultaneous speech streams are assessed, reflecting the reality of hearing in the everyday world. Qualities of hearing experience include ease of listening, and the naturalness, clarity and identifiability of different speakers, different musical pieces and instruments, and different everyday sounds. Application of the SSQ to 153 new clinic clients prior to hearing aid fitting showed that the greatest difficulty was experienced with simultaneous speech streams, ease of listening, listening in groups and in noise, and judging distance and movement. SSQ ratings were compared with an independent measure of handicap. After differences in hearing level were controlled for, it was found that identification, attention and effort problems, as well as spatial hearing problems, feature prominently in the disability handicap relationship,. along with certain features of speech hearing. The results implicate aspects of temporal and spatial dynamics of hearing disability in the experience of handicap. The SSQ shows promise as an instrument for evaluating interventions of various kinds, particularly (but not exclusively) those that implicate binaural function.

Aged↗

PubMed2001

Position-specific and non-colinear expression of the planarian posterior (Abdominal-B-like) gene.

Hox genes are pivotal molecules in the control of morphogenesis along the anterior-posterior (AP) axis in various bilaterians. Planarians are key animals for understanding the evolution of the bilaterian body plan. Furthermore, they are also known for their strong regeneration ability and are thought to use the Hox genes in the process of reconstruction of the AP axis. In the present paper, the identification and analysis of expression of two posterior (Abdominal-B-like) genes, DjAbd-Ba and DjAbd-Bb, is reported in the planarian Dugesia japonica. DjAbd-Ba is expressed in the entire tail region and its anterior boundary is the posterior pharyngeal region. In contrast, DjAbd-Bb is expressed in several types of cells throughout the body. During regeneration, the expression of DjAbd-Ba rapidly recovers a pattern similar to that in the normal worm. These findings suggest the possibility that DjAbd-Ba is involved in the specification of the tail region. The anterior boundary of the expression domain of the posterior gene DjAbd-Ba is anterior to the domains of the central genes Plox4-Dj and Plox5-Dj. These expression patterns of planarian Hox genes seem out of the rule of spatial colinearity and may reflect an ancestral feature of bilaterian Hox genes.

Amino Acid Sequence↗

PubMed1995

Solution structure of the Ras-binding domain of c-Raf-1 and identification of its Ras interaction surface.

The structure of the Ras-binding domain of human c-Raf-1 (residues 55-132) has been determined in solution by nuclear magnetic resonance (NMR) spectroscopy. Following complete assignment of the backbone and side-chain 1H, 15N, and 13C resonances, the structure was calculated using the program CHARMM. Over 1300 NOE-derived constraints were applied, resulting in a detailed structure. The fold of Raf55-132 consists of a five-stranded beta-sheet, a 12-residue alpha-helix, and an additional one-turn helix. It is similar to those of ubiquitin and the IgG-binding domain of protein G, although the three proteins share very little sequence identity. The surface of Raf55-132 that interacts with Ras has been identified by monitoring perturbation of line widths and chemical shifts of 15N-labeled Raf55-132 resonances during titration with unlabeled Ras-GMPPNP. The Ras-binding site is contained within a spatially contiguous patch comprised of the N-terminal beta-hairpin and the C-terminal end of the alpha-helix.

Binding Sites↗

PubMed2002

CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains.

CENP-C is a fundamental component of the centromere, highly conserved among species and necessary for the proper assembly of the kinetochore structure and for the metaphase-anaphase transition. Although CENP-C can bind DNA in vitro, the identification of the DNA sequences associated with it in vivo and the significance of such an interaction have been, until now, elusive. To address this problem we took advantage of a chromatin-immunoprecipitation procedure and applied this technique to human HeLa cells. Through this approach we could establish that: (1) CENP-C binds the alpha-satellite DNA selectively; (2) the CENP-C region between amino acids 410 and 537, previously supposed to contain a DNA-binding domain, is indeed required to perform such a function in vivo; and (3) the profile of the alpha-satellite DNA associated with CENP-C is essentially identical to that recognized by CENP-B. However, further biochemical and ultrastructural characterization of CENP-B/DNA and CENP-C/DNA complexes, relative to their DNA components and specific spatial distribution in interphase nuclei, surprisingly reveals that CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains. Our results, besides extending previous observations on the role of CENP-C in the formation of active centromeres, show, for the first time, that CENP-C can associate with the centromeric DNA sequences in vivo and, together with CENP-B, defines a highly structured organization of the alpha-satellite DNA within the human centromere.

Autoantigens↗

PubMed2005

Homology modelling of transferrin-binding protein A from Neisseria meningitidis.

Neisseria meningitidis, a causative agent of bacterial meningitis, obtains transferrin-bound iron by expressing two outer membrane located transferrin-binding proteins, TbpA and TbpB. TbpA is thought to be an integral outer membrane pore that facilitates iron uptake. Evidence suggests that TbpA is a useful antigen for inclusion in a vaccine effective against meningococcal disease, hence the identification of regions involved in ligand binding is of paramount importance to design strategies to block uptake of iron. The protein shares sequence and functional similarities to the Escherichia coli siderophore receptors FepA and FhuA, whose structures have been determined. These receptors are composed of two domains, a 22-stranded beta-barrel and an N-terminal plug region that sits within the barrel and occludes the transmembrane pore. A three-dimensional TbpA model was constructed using FepA and FhuA structural templates, hydrophobicity analysis and homology modelling. TbpA was found to possess a similar architecture to the siderophore receptors. In addition to providing insights into the highly immunogenic nature of TbpA and allowing the prediction of potentially important ligand-binding epitopes, the model also reveals a narrow channel through its entire length. The relevance of this channel and the spatial arrangement of external loops, to the mechanism of iron translocation employed by TbpA is discussed.

Amino Acid Sequence↗

PubMed2004

Identification of 51 novel exons of the Usher syndrome type 2A (USH2A) gene that encode multiple conserved functional domains and that are mutated in patients with Usher syndrome type II.

The USH2A gene is mutated in patients with Usher syndrome type IIa, which is the most common subtype of Usher syndrome and is characterized by hearing loss and retinitis pigmentosa. Since mutation analysis by DNA sequencing of exons 1-21 revealed only ~63% of the expected USH2A mutations, we searched for so-far-uncharacterized exons of the gene. We identified 51 novel exons at the 3' end of the gene, and we obtained indications for alternative splicing. The putative protein encoded by the longest open reading frame harbors, in addition to the known functional domains, two laminin G and 28 fibronectin type III repeats, as well as a transmembrane region followed by an intracellular domain with a PDZ-binding domain at its C-terminal end. Semiquantitative expression profile analysis suggested a low level of expression for both the long and the short isoform(s) and partial overlap in spatial and temporal expression patterns. Mutation analysis in 12 unrelated patients with Usher syndrome, each with one mutation in exons 1-21, revealed three different truncating mutations in four patients and two missense mutations in one patient. The presence of pathogenic mutations in the novel exons indicates that at least one of the putative long isoforms of the USH2A protein plays a role in both hearing and vision.

Alternative Splicing↗

PubMed2000

[The identification and cloning of human ubiquitin binding enzyme cDNA].

OBJECTIVE: To identify and clone the gene encoding human ubiquitin binding enzyme and study its expression spectrum. METHODS: According to the sequence of human EST, which is highly homologous to the mouse ubiquitin conjugating enzyme (E2), primers used for library screening were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by making use of bioinformatics and its expression spectrum was studied by using multiple-tissue Northern blot. RESULTS: Two cDNA clones encoding human ubiquitin conjugating enzyme were isolated and identified. Both containing the ubiquitin conjugating domain, they were 88% identical in amino acid sequences and found to be isoforms of each other with only an exon excised from the short sequence. They belonged to a highly conserved, and widely expressed E2 enzyme family. Northern blot showed that they were expressed exclusively in heart, placenta, and pancreas while no transcripts could be detected in brain, lung, liver, skeletal muscle, or kidney. CONCLUSIONS: The gene encoding human ubiquitin binding enzyme is expressed under both temporal and spatial control. As a key enzyme in the degradation of proteins, ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post-translation.

Amino Acid Sequence↗

PubMed2007

Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites.

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3-17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

Amino Acid Sequence↗

PubMed1999

Cloning of two novel mammalian paralogs of relaxin/insulin family proteins and their expression in testis and kidney.

Based on sequence homology to insulin and relaxin, we have isolated two novel genes of the insulin superfamily from mouse tissues. Because these proteins show a high similarity to relaxin and relaxin-like factor (RLF or Ley I-L), they were named as RIF1 (relaxin/insulin-like factor 1) and RIF2 (relaxin/insulin-like factor 2). After RT-PCR, full-length cDNAs of RIF1 and RIF2 were obtained from mouse testis and ovary, respectively. In addition, a putative human ortholog of RIF1 was isolated from human testis. The deduced coding regions of mRIF1, mRIF2, and hRIF1 were 191, 145, and 213 amino acids, respectively, and all three proteins contain a typical signal sequence for secretion at their amino terminus. Sequence comparison indicated that RIFs encode proteins consisting of B and A subunits connected by a long C domain peptide, and the deduced mature proteins of these putative ligands are most closely related to relaxin, RLF, and insulin from different species. Northern blot analysis showed that RIF1 transcripts are approximately 1.2 kb in size and are expressed mainly in testis of mouse and human. In contrast, RIF2 message of 2.0 and 1.2 kb are preferentially expressed in mouse kidney and are lower in testis, heart, and brain. In addition, immunohistochemical analysis showed that testis expression of RIF1 is restricted to interstitial cells surrounding seminiferous tubules. In kidney, the RIF2 message is localized to selected epithelial cells of loop of Henle. The exclusive expression pattern of RIF1 and related RLF in testis interstitial cells suggested potential physiological roles of these two distinct insulin/relaxin family ligands in testis function. Additionally, the spatial expression pattern of RIF2 suggests a novel role of RIF2 in nephrophysiology. Identification of RIF polypeptides expands the family of relaxin- and insulin-like hormones and allows future elucidation of the physiological role and hormonal mechanisms for these tissue-specific factors.

Amino Acid Sequence↗

PubMed2006

Detection of emotional expressions in rapidly changing facial displays in high- and low-socially anxious women.

Facial information and attention to facial displays are distributed over spatial as well as temporal domains. Thus far, research on selective attention to (dis)approving faces in the context of social anxiety has concentrated primarily on the spatial domain. Using a rapid serial visual presentation (RSVP) paradigm, the present study examined the temporal characteristics of visual attention for happy and angry faces in high- (n=16) and low-socially anxious individuals (n=17), to test whether also in the temporal domain socially anxious individuals are characterized by threat-confirming attentional biases. Results indicated that presenting angry faces as the first target (T1) did not aggravate the detection of the emotional expression of the second target (T2). Yet, participants generally showed superior detection of the emotional expression of T2, if T2 was an angry face. Casting doubt on the role of such attenuated attentional blink for angry faces in social anxiety, no evidence emerged to indicate that this effect was relatively strong in high-socially anxious individuals. Finally, the presentation of an angry face as T2 resulted in a relatively hampered identification of a happy-T1. Again, this "backward blink" was not especially pronounced in high-socially anxious individuals. The present anger superiority effects are consistent with evolutionary models stressing the importance of being especially vigilant for signals of dominance. Since the effects were not especially pronounced in high-anxious individuals, the present study adds to previous findings indicating that socially anxious individuals are not characterized by a bias in the (explicit) detection of emotional expressions [Philippot, P., & Douilliez, C. (2005). Social phobics do not misinterpret facial expression of emotion. Behaviour Research and Therapy, 43, 639-652].

Adolescent↗

PubMed2005

Identification of a hypaxial somite enhancer element regulating Pax3 expression in migrating myoblasts and characterization of hypaxial muscle Cre transgenic mice.

Pax3 encodes a transcription factor that functions in the embryonic central nervous system, neural crest, and somitic mesoderm. Prior studies suggest that distinct regulatory elements regulate temporal and spatial expression of Pax3 in neural crest and mesoderm. Here, we describe a discrete enhancer element, conserved between mouse and human genomes, that directs Pax3 expression in the ventral-lateral lip of interlimb somites. These regions give rise to hypaxial musculature including limb, ventral body wall, diaphragm, and tongue muscles. Transgenic mice harboring the hypaxial muscle enhancer driving lacZ expression initiate beta-galactosidase expression at E10.0, significantly later than endogenous Pax3 expression in presomitic and segmented mesoderm. Initiation of transgene expression is not dependent on Pax3 itself, since expression is detectable in homozygous Splotch embryos. Transgenic mice expressing Cre recombinase in hypaxial myoblasts were generated and characterized. These results suggest that Pax3 is differentially regulated within the somite in both spatial and temporal domains. Hypaxial muscle Cre mice will allow for specific manipulation of gene expression in this subset of developing skeletal muscle.

Animals↗

PubMed2001

Mutational analysis of the K+-competitive inhibitor site of gastric H,K-ATPase.

The gastric H,K-ATPase is inhibited selectively and K(+)-competitively from its luminal surface by protonated imidazo[1,2alpha]pyridines (e.g., SCH28080). Identification of the amino acids in the membrane domain that affect SCH28080 inhibition should provide a template for modeling a luminally directed vestibule in this enzyme, based on the crystal structure of the sr Ca-ATPase. Five conserved carboxylic residues, Glu343, Glu795, Glu820, Asp824, Glu936, and unique Lys791 in the H,K-ATPase were mutated, and the effects of mutations on the K(i) for SCH28080, V(max), and K(m,app)[NH(4)(+)] were measured. A kinetic analysis of the ATP hydrolysis data indicated that all of these residues significantly affect the interaction of NH(4)(+) ions with the protein but only three of them, Glu795, Glu936, and Lys791, greatly affected SCH28080 inhibition. A Glu795Asp mutation increased the K(i) from 64 +/- 11 to 700 +/- 110 nM. Since, however, the mutation Glu795Gln did not change the K(i) (86 +/- 31 nM), this site has a significant spatial effect on inhibitor kinetics. A Glu936Asp mutation resulted in noncompetitive kinetics while Gln substitution had no effect either on inhibitor affinity or on the nature of the kinetics, suggesting that the length of the Glu936 side chain is critical for the exclusive binding of the ion and SCH28080. Mutation of Lys791 to Ser, the residue present in the SCH28080-insensitive Na,K-ATPase, resulted in a 20-fold decrease in SCH28080 affinity, suggesting an important role of this residue in SCH28080 selectivity of the H,K-ATPase versus Na,K-ATPase. Mutations of Asp824, Glu343, and Glu820 increased the K(i) 2-3-fold, implying a relatively minor role for these residues in SCH28080 inhibition. It appears that the imidazopyridine moiety of SCH28080 in the protonated state interacts with residues near the negatively charged residues of the empty ion site from the luminal side (TM4, -5, -6, and -8) while the hydrophobic phenyl ring interacts with TM1 or TM2 (the latter conclusion based on previous data from photoaffinity labeling). The integrity of the SCH28080 binding site depends on the presence of Lys791, Glu936, and Glu795 in H,K-ATPase. A computer-generated model of this region illustrates the possible involvement of the residues previously shown to affect SCH28080 inhibition (Cys813, Ile816, Thr823, Met334, Val337) and may predict other residues that line the SCH28080 binding vestibule in the E(2) conformation of the pump.

Amino Acid Sequence↗

PubMed2006

Cross genome comparisons of serine proteases in Arabidopsis and rice.

BACKGROUND: Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa) genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. RESULTS: Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively). Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. CONCLUSION: The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

Amino Acid Sequence↗

PubMed2002

Binding of cGMP to GAF domains in amphibian rod photoreceptor cGMP phosphodiesterase (PDE). Identification of GAF domains in PDE alphabeta subunits and distinct domains in the PDE gamma subunit involved in stimulation of cGMP binding to GAF domains.

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Palphabeta) and two identical regulatory subunits (Pgamma). Biochemical studies have shown that amphibian Palphabeta has high affinity, cGMP-specific, non-catalytic binding sites and that Pgamma stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pgamma-depleted membrane-bound PDE (20-40% Pgamma still present), a single type of cGMP-binding site with a relatively low affinity (K(d) approximately 100 nm) was observed, and addition of Pgamma increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pgamma reduced its ability to stimulate cGMP binding. Among these, the site involved in Pgamma phosphorylation by Cdk5 (positions 20-23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pgamma inhibition of the cGMP hydrolytic activity of Palphabeta. In addition, the Pgamma concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Palphabeta heterodimer contains two spatially and functionally distinct types of Pgamma-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Palphabeta-Pgamma interaction in which Pgamma, by binding to one of the two sites in Palphabeta, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.

Amino Acid Sequence↗