A novel isoform of the mitochondrial outer membrane protein VDAC3 via alternative splicing of a 3-base exon. Functional characteristics and subcellular localization

Voltage-dependent anion channels (VDACs) are pore-forming proteins found in the outer mitochondrial membrane of all eucaryotes. VDACs are the major pathway for metabolites through the outer mitochondrial membrane and, in mammals, bind several cytosolic carbohydrate kinases. Whereas yeast contain a single VDAC (YVDAC), to date three isoforms have been described in the mouse that constitute a gene family. We have observed an additional isoform of VDAC3 that appears to be generated via the tissue-specific alternative splicing of a 3-base exon (ATG). The exon is predicted to introduce a methionine 39 amino acids downstream of the amino terminus of the polypeptide. Between exons 3 and 4 is an intronic sequence that potentially encodes the exon, with flanking splice enhancer elements. Expression of this alternative form in the mouse is limited to brain, heart, and skeletal muscle. Complementation of YVDAC-deficient yeast by the two isoforms and with other sequence variants of VDAC3 suggests this residue is an important modulator of VDAC3 function. In transfected mammalian cells both isoforms localize to mitochondria. A similar variant is present in humans.     

Molecular cloning of two isoforms of the guinea pig C3a anaphylatoxin receptor: alternative splicing in the large extracellular loop

The anaphylatoxin C3a is released from C3 during complement activation. C3a is a potent spasmogen and has recently been described as an eosinophil and mast cell chemotactic factor that mediates a number of inflammatory reactions. Previously, we demonstrated the presence of a specific C3a receptor (C3aR) on guinea pig platelets. We report here the isolation of cDNA clones encoding for two isoforms of guinea pig C3aR (gpC3aR). Hydropathy analysis of the deduced amino acid sequence of both gpC3aR clones indicated seven transmembrane domains with a large extracellular (EC) loop between the fourth and fifth transmembrane domains, which is a known characteristic of the human C3aR. Northern blot analysis revealed that the gpC3aR was abundantly expressed on macrophages and in the spleen. A comparison of the deduced amino acid sequence of the larger gpC3aR (gpC3aR-L) with the recently cloned human C3aR indicated a 59.5% identity. The deduced amino acid sequence of the second, smaller cDNA clone was identical with gpC3aR-L, except that it lacked 35 amino acids in the large EC loop. Our evidence indicates that alternative splicing occurred in the large EC loop that accounts for these two isoforms. L cells separately expressing one of these two isoforms of the gpC3aR showed similar high-affinity C3a binding. An RT-PCR analysis documented that both forms of the C3aR were expressed in a variety of guinea pig tissues. The cloning and expression of these two natural forms of gpC3aR cDNA indicated that the deletion of the 35-residue portion of the large EC loop of gpC3aR-L did not alter C3a binding.     

Fused aminotetralins: novel antagonists with high selectivity for the dopamine D3 receptor

Starting from a series of 2-aminotetralins 1, a novel series of N-[4-(4-phenylbenzoylamino)butyl]-octahydrobenzoquinolines and hexahydrobenzoindoles with high potency and selectivity for the dopamine D3 receptor has been designed. The effect of ligand chirality on binding affinity has been established. Selected derivatives (e.g. 2o, 2p) show high functional selectivity and enhanced in vivo properties compared to 1.     

SAR of novel biarylmethylamine dopamine D4 receptor ligands

SAR for a novel series of dopamine D4 receptor ligands is shown. Very selective, highly potent compounds like 1-(2-pyrimidinyl)-4-(3-(3-thienyl)-benzyl)-piperazine (5f) and 2-(4-(1-fluorenylmethyl)-1-piperazinyl)-pyrimidine (8c) were obtained.     

A role for amino acid residues in the third cytoplasmic loop in defining the ligand binding characteristics of the alpha2D-adrenergic receptor

In this report, 5 amino acid residues (aa) in the third cytoplasmic loop of the alpha 2D-adrenergic receptor are identified which (individually or together) alter its ligand-binding characteristics. An important structural discrepancy exists in the third cytoplasmic loop of the alpha 2D-ARs encoded by the rat cDNA and the rat gene--five aa are different. The newly identified bovine receptor as well as the mouse receptor contained the 5 aa identical to that encoded by the rat cDNA. Site-directed mutation of these residues to those of the rat gene encoded receptor resulted in alteration of binding characteristics: significant changes in the ability of the mutant receptor to bind to a number of agonists and antagonists were observed--ranging from a decrease by half in the case of oxymetazoline, to near total loss of binding in the case of prazosin. Thus, the mutant receptor was no longer pure alpha 2D-AR. This indicated a hitherto unrealized role of the third cytoplasmic loop in defining the ligand-binding characteristics of the receptor, and also suggested that the rat gene sequence was most probably in error.     

SH3 binding domains in the dopamine D4 receptor

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.     

Truncated human endothelin receptor A produced by alternative splicing and its expression in melanoma

PURPOSE: Traumatic sphincter disruption frequently is associated with a rectovaginal fistula, but the effect of a persistent sphincter defect on the outcome of rectovaginal fistula repair is poorly documented. We analyzed the outcome of rectovaginal fistula repairs based on preoperative sphincter status. PATIENTS AND METHODS: We identified 52 women who underwent 62 repairs of simple obstetrical rectovaginal fistulas between 1992 and 1995. Fourteen patients (27 percent) had preoperative endoanal ultrasound studies and 25 (48 percent) had anal manometry studies. Follow-up was by mailed questionnaire in 36 patients (69 percent) and by telephone interview in 12 (23 percent), for a total response rate of 92 percent. Median age was 30.5 (range, 18-70) years, and median follow-up was 15 (range, 0.5-123) months. Twenty-five patients (48 percent) complained of varying degrees of fecal incontinence before surgery. There were 27 endorectal advancement flaps and 35 sphincteroplasties (28 with and 8 without levatoroplasty). RESULTS: Success rates were 41 percent with endorectal advancement flaps and 80 percent with sphincteroplasties (96 percent success with and 33 percent without levatoroplasty; P = 0.0001). Endorectal advancement flap was successful in 50 percent of patients with normal sphincter function but in only 33 percent of patients with abnormal sphincter function (P = not significant). For sphincteroplasties, success rates were 73 vs. 84 percent for normal and abnormal sphincter function, respectively (P = not significant). Results were better after sphincteroplasties vs. endorectal advancement flaps in patients with sphincter defects identified by endoanal ultrasound (88 vs. 33 percent; P = not significant) and by manometry (86 vs. 33 percent; P = not significant). Poor results correlated with prior surgery in patients undergoing endorectal advancement flaps (45 percent vs. 25 percent; P = not significant) but not sphincteroplasties (80 vs. 75 percent; P = not significant). CONCLUSIONS: All patients with rectovaginal fistula should undergo preoperative evaluation for occult sphincter defects by endoanal ultrasound or anal manometry or both procedures. Local tissues are inadequate for endorectal advancement flap repairs in patients with sphincter defects and a history of previous repairs. Patients with clinical or anatomic sphincter defects should be treated by sphincteroplasty with levatoroplasty.     

Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain

The NMDA (N-methyl D-aspartate) receptors in the brain play a critical role in synaptic plasticity, synaptogenesis and excitotoxicity. Molecular cloning has demonstrated that NMDA receptors consist of several homologous subunits (NMDAR1, 2A-2D). A variety of studies have suggested that protein phosphorylation of NMDA receptors may regulate their function and play a role in many forms of synaptic plasticity such as long-term potentiation. We have examined the phosphorylation of the NMDA receptor subunit NMDAR1 (NR1) by protein kinase C (PKC) in cells transiently expressing recombinant NR1 and in primary cultures of cortical neurons. PKC phosphorylation occurs on several distinct sites on the NR1 subunit. Most of these sites are contained within a single alternatively spliced exon in the C-terminal domain, which has previously been proposed to be on the extracellular side of the membrane. These results demonstrate that alternative splicing of the NR1 messenger RNA regulates its phosphorylation by PKC, and that mRNA splicing is a novel mechanism for regulating the sensitivity of glutamate receptors to protein phosphorylation. These results also provide evidence that the C-terminal domain of the NR1 protein is located intracellularly, suggesting that the proposed transmembrane topology model for glutamate receptors may be incorrect.     

Alterations in dopamine release but not dopamine autoreceptor function in dopamine D3 receptor mutant mice

Dopamine (DA) autoreceptors expressed along the somatodendritic extent of midbrain DA neurons modulate impulse activity, whereas those expressed at DA nerve terminals regulate both DA synthesis and release. Considerable evidence has indicated that these DA autoreceptors are of the D2 subtype of DA receptors. However, many pharmacological studies have suggested an autoreceptor role for the DA D3 receptor. This possibility was tested with mice lacking the D3 receptor as a result of gene targeting. The basal firing rates of DA neurons within both the substantia nigra and ventral tegmental area were not different in D3 receptor mutant and wild-type mice. The putative D3 receptor-selective agonist R(+)-trans-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-(1)benzopyrano(4,3-b)-1,4-oxazin+ ++-9-ol (PD 128907) was equipotent at inhibiting the activity of both populations of midbrain DA neurons in the two groups of mice. In the gamma-butyrolactone (GBL) model of DA autoreceptor function, mutant and wild-type mice were identical with respect to striatal DA synthesis and its suppression by PD 128907. In vivo microdialysis studies of DA release in ventral striatum revealed higher basal levels of extracellular DA in mutant mice but similar inhibitory effects of PD 128907 in mutant and wild-type mice. These results suggest that the effects of PD 128907 on dopamine cell function reflect stimulation of D2 as opposed to D3 receptors. Although D3 receptors do not seem to be significantly involved in DA autoreceptor function, they may participate in postsynaptically activated short-loop feedback modulation of DA release.     

Functional implications of multiple dopamine receptor subtypes: the D1/D3 receptor coexistence

The D3 dopamine receptor, a D2-like receptor, is selectively expressed in the ventral striatum, particularly in the shell of nucleus accumbens and islands of Calleja, where it is found in medium sized substance P neurons. The latter co-express the D1 receptor whose interaction with the D3 receptor was studied by treating rats with selective agonists and antagonists. In agreement with the opposite cAMP response, they mediate in cultured neuroblastoma cells, the D1 and D3 receptors exerted opposite influences on c-fos expression in islands of Calleja. However, in agreement with the synergistic influence of cAMP on D3 receptor-mediated mitogenesis on the same cultured cells, D1 and D3 receptor stimulation in vivo synergistically enhanced preprotachykinin mRNA in the shell of accumbens. This indicates that the two receptor subtypes may affect neurons in either synergy or opposition according to the cell or signal generated. Levodopa-induced behavioral sensitization in hemiparkinsonian rats is another example of D1/D3 receptor interaction. Hence repeated levodopa administration induces the ectopic appearance of the D3 receptor in substance P/dynorphin, striatonigral neurons of the dorsal striatum. This induction is secondary to D1 receptor stimulation in neurons of the denervated side and fully accounts for the sensitization, i.e. the increased behavioral responsiveness to levodopa. During brain development, a similar process could operate to control the late appearance of the D3 receptor in D1-receptor bearing neurons of the ventral striatum at a time at which they start to be innervated by dopamine neurons. Finally, taking into account a variety of genetic, developmental, neuroimaging and pharmacological data, we postulate that imbalances between the levels of D1 and D3 receptors in the same neurons could be responsible for schizophrenic disorders.     

Characterization of protein kinase C-mediated phosphorylation of the short cytoplasmic domain isoform of C-CAM

BACKGROUND: Picture Archiving Communication System (PACS) is a sophisticated software and hardware package that enables clinicians to retrieve, review, and digitally manipulate radiographs from computer workstations throughout the hospital. PACS was instituted at Brooke Army Medical Center in July 1993. METHODS: Fifty consecutive trauma and 50 consecutive motor vehicle crash (MVC) trauma admissions to an urban trauma center were reviewed before PACS (January 1993) and 18 months after PACS was instituted (January 1995). Patients were compared by the number of radiographs needed during the initial evaluation by type and total. The trauma groups were subdivided by mechanism and also compared. Demographic and physiologic data were collected for each patient. RESULTS: There are no differences in the demographic and physiologic data between groups. For the 50 consecutive trauma admissions, only two areas of statistical difference were found: more chest films were obtained in the MVC PACS group and more pelvis films were obtained in the gunshot wound pre-PACS group. For the 50 consecutive MVC trauma admissions, the PACS group had more chest and total radiographs per patient than the pre-PACS group. More computed tomographic scans of the neck were obtained in the PACS group. CONCLUSION: PACS did not decrease the number of radiographs needed to adequately and fully evaluate the trauma patient.     

Structure of two fragments of the third cytoplasmic loop of the rat angiotensin II AT1A receptor. Implications with respect to receptor activation and G-protein selection and coupling

Managed care is an increasingly widespread way of delivering health care that combines medical care provider systems (doctors, nurses, clinics, hospitals) with the payor system (health insurance). This article gives a historical overview of managed health care, discusses managed HIV care, and managed care reform.     

Imaging D2 receptor occupancy by endogenous dopamine in humans

The impact of endogenous dopamine on in vivo measurement of D2 receptors in humans was evaluated with single photon emission computerized tomography (SPECT) by comparing the binding potential (BP) of the selective D2 radiotracer [123I]IBZM before and after acute dopamine depletion. Dopamine depletion was achieved by administration of the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT), given orally at a dose of 1 g every six hours for two days. AMPT increased [123I]IBZM BP by 28 +/- 16% (+/- SD, n = 9). Experiments in rodents suggested that this effect was due to removal of endogenous dopamine rather than D2 receptor upregulation. Synaptic dopamine concentration was estimated as 45 +/- 25 nM, in agreement with values reported in rodents. The amplitude and the variability of the AMPT effect suggested that competition by endogenous dopamine introduces a significant error in measurement of D2 receptors in vivo with positron emission tomography (PET) or SPECT. However, these results also imply that D2 receptor imaging coupled with acute dopamine depletion might provide estimates of synaptic dopamine concentration in the living human brain.     

Deletions in the third intracellular loop of the thyrotropin receptor. A new mechanism for constitutive activation

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.     

Preferential involvement of D3 versus D2 dopamine receptors in the effects of dopamine receptor ligands on oral ethanol self-administration in rats

There is evidence that dopamine transmission is involved in reinforcement processes and the present study investigated the relative involvement of D3 versus D2 dopamine receptors in the effects of dopamine ligands on the reinforcing action of ethanol. Rats were trained to self-administer ethanol (10% v/v) orally in a free-choice two-lever operant task using a saccharin-fading procedure. When preference in responding for ethanol over water had developed the rats were tested with several dopamine agonists and antagonists. Pretreatment with the non-selective dopamine agonist, apomorphine (0.01-0.1 mg/kg), the preferential D2 agonist, bromocriptine (1-10 mg/kg) and the selective D3 agonists, 7-OH-DPAT (0.003-0.1 mg/kg), PD 128907 (0.1-3 mg/kg), (+)3PPP (0.3-3 mg/kg), quinelorane (0.0001-0.003 mg/kg) and quinpirole (0.003-0.03 mg/kg), resulted in dose-dependent decreases in responding for ethanol. The relative potencies of the dopamine agonists to decrease ethanol self-administration were highly correlated with their published potencies to produce in vitro functional D3 but not D2 responses. Active doses could be considered as those selectively stimulating receptors involved in the control of dopamine release, suggesting that reduction of dopamine transmission was associated with a decrease in ethanol-reinforced responding. This conclusion was further supported by the finding that pretreatment with the D2/D3 dopamine antagonists, haloperidol (0.1-0.4 mg/kg) and tiapride (10-60 mg/kg), decreased responding for ethanol at doses which have been shown previously to block dopamine transmission.