Secretagogue Effects on G-Protein mRNA Levels in ACTH/Endorphin-Producing Pituitary Cells E.A. Thiele, R.R. Reed, and B.A. Eipper Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Guanine nucleotide regulatory proteins (G proteins) play an important role in signal transduction in many systems, and are best described in the adenylate cyclase system. We find significant alterations in G protein mRNA levels and protein levels in AtT-20 cells in response to agents that affect secretion by modulating adenylate cyclase activity. These results suggest that regulation of G protein levels may be an important mechanism in determining the responsiveness of a cell to stimuli. In AtT-20/D16V mouse corticotrope tumor cells, both Gs and G1 are involved in regulating secretion of proACTH/endorphin (PAE): Corticotropin releasing factor (CRF) stimulates the secretion of PAE by stimulating adenylate cyclase activity through Gs, and somatostatin inhibits PAE secretion by inhibiting adenylate cyclase activity through Gj. Pretreatment with dexamethasone, a synthetic glucocorticoid, suppresses the CRF stimulated secretion. AtT-20 cells were treated with 100 nM CRF or 100 nM somatostatin for 6 hours, or with 1 μM dexamethasone for 72 hours. Radioimmunoassay showed that the treatments had the expected results on peptide secretion. The cDNAs corresponding to Gas, Gai, and GB (Jones and Reed, J. Biol. Chem. 262:14241-14249, 1987) were used to probe Northern blots of total cellular RNA from AtT-20 cells. Northern analysis indicated significant increases in the levels of mRNA for all three G protein subunits following treatment with CRF, somatostatin, and dexamethasone. Surprisingly, treatment with both CRF and somatostatin resulted in either no change or a decrease in mRNA levels for the G protein subunits. Protein levels for the Ga and GB subunits were also affected by the various treatments, as determined by Western analysis. VAMP-1: A Synaptic Vesicle-Associated William S. Trimble, David M. Cowan, and Richard H. Scheller Several proteins are associated with, or integral components of, the lipid bilayer which forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we have used antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a lambda gt11 expression library constructed from mRNA of the electromotor nucleus. One of the clones isolated in this screen encodes a protein which we have called VAMP-1 for vesicle-associated membrane protein-1. The nucleotide sequence of this and two longer clones was determined. An open reading frame of 120 amino acids encodes a protein with a predicted molecular weight of 13.0 kD whose primary sequence can be divided into three domains: a proline rich amino terminus, a highly charged internal region, and a hydrophobic carboxyterminal domain which is predicted to comprise a membrane anchor. The gene for VAMP-1 is expressed as a 2.5 kb transcript only in the electric lobe and brain. Antiserum raised against a VAMP-1/B-gal fusion protein demonstrated the presence of a 17 kD protein in the brain, electric lobe, and the electric organ, and this antigen is specifically enriched during vesicle purification. Tryptic digestion experiments show that the antigen is exposed on the cytoplasmic side of the vesicles. The nucleotide sequence of a VAMP-1 related cDNA clone from the rat was found to share greater than 80% amino acid homology with most extensive homology occurring in the charged internal domain. These data suggest that VAMP-1 is an evolutionarily conserved integral membrane protein on the cytoplasmic side of synaptic vesicles where it may play a role in packaging, transport, or release of neurotransmitters. Human Postmortem Brain mRNA'S: 'In Situ' Hybridization of CRNA Probe for Neuropeptide Y in Rat and Human Brain J. P. Vonsattel, J.M. Allen, and E.D. Bird Brain Tissue Resource Center, McLean Hospital and Massachusetts General Hospital/Harvard Medical School, Belmont, MA 02178 Since valuable brain tissues suitable for mRNA studies often become available to the Brain Tissue Resource Center, a practical, simple, standardized method of preserving the brain needs to be established. Northern blot analysis using probes for ẞ amyloid and GAP-43 protein reveals that the degree of mRNA degradation in various human brains studied did not correlate with the time of brain removal after death. A human and a rat 35s-labeled cRNA probe for neuropeptide Y (NPY) was used in both a human (postmortem delay, 5 hours) and a perfused rat brain in order to assess the retention of the corresponding mRNA by 'in situ' hybridization. The human brain was removed from the skull 4 hours after death, and the hypothalamus was immersed 1 hour later in fresh 4% paraformaldehyde (pH 7.6) and kept overnight at 4°C. The rat was perfused with the same fixative; then the brain was removed from the skull and processed in the same way as the human hypothalamus. Prehybridization, hybridization, and autoradiographic detection of multiple samples of both human and rat specimens proceeded simultaneously in the same conditions. Occasional discrete cell resolutions of the human NPY cRNA hybridizing signal were present in the subependymal region about the third ventricle dorsal to the mammillary body. There was an absence of signal in serial sections to which no probe was applied and to which the rat NPY cRNA probe was applied. Similarly, rat hypothalamic coronal section to which rat NPY cRNA was applied yielded discrete increased autoradiographic grain density over cells in the arcuate nucleus. Serial sections, which did not receive the probe and which received human NPY CRNA, were devoid of specific hybridization. This preliminary study reveals that the mRNA for NPY is well retained in the human hypothalamus with a postmortem delay of 5 hours, and it appears that the probes were species specific. (Supported by The Wills Foundation.) Autoregulation of CA++/Calmodulin-Dependent M. Neal Waxham and Paul T. Kelly The University of Texas Medical School in Houston Recent studies have shown that the calmodulin (CaM)-binding domain of CK-II resides between two consensus phosphorylation sequences, Arg-HisGlu-Thr and Arg-Asn-Phe-Ser at the N- and C-terminus, respectively. CK-II activity is initially dependent on Ca++ and CaM; however, following autophosphorylation, its activity becomes independent of Ca++/CaM. Experimental evidence suggest that the phosphorylation of a Thr residue(s) procedures the Ca++/CaM-independent form of CK-II. We report here that a synthetic peptide containing the CaM-binding domain and the consensus phosphorylation sequence at its N-terminus contains bifunctional inhibitory properties. As expected, the peptide acts as an antagonist (IC50 = 75 nM) of Ca++/CaM-dependent activation of CK-II by competing for Ca++/CaM. However, we found that the peptide also inhibits (IC50 = 2.5 μ) synapsin I phosphorylation and further autophosphorylation using a form of CK-II that is independent of Ca++/CaM for activity. A second peptide, identical to the one just described but lacking the arginine residue necessary to form the N-terminal phosphorylation sequence, exhibits similar Ca++/CaM_antagonist properties, but did not inhibit Ca++/CaM-independent CK-II activity. These results suggest that the sequence just N-terminal to the CaM-binding domain of CK-II can bind to the enzyme's active site and inhibit activity. We suggest that this active-site directed inhibitory domain naturally resides in the active site of the nonphosphorylated, Ca++/CaM-dependent enzyme. Binding of Ca++/CaM and subsequent phosphorylation of the N-terminal autophosphorylation sequence causes a disinhibition of the active site and permits further substrate phosphorylation. Whether or not phosphorylation of this site is important in the generation of Ca++/CaM-independent CK-II activity awaits further investigation. Multiple Mechanisms of Regulation of Sherwin Wilk and Chen-Shian Suen Department of Pharmacology, Mount Sinai School of Medicine, The biological activity of neuropeptides may be controlled in part by the enzymes catalyzing their degradation. Pyroglutamyl peptidase I (EC3.4.19.3), a cytosolic cysteine protease degrades TRH, LHRH, neurotensin, and bombesin. Pyroglutamyl peptidase II (EC 3.4.19.-), a synaptosomal membranebound metallo-protease appears to be specific for TRH. We provide evidence for regulation of both enzymes in cell cultures and in vivo. The activity of pyroglutamyl peptidase I in GH3 cells is increased when cells are grown in the presence of 5-oxoprolinal, a specific pyroglutamyl peptidase I inhibitor. The activity of this enzyme in GH3 cells is also increased by low concentrations of L-3,5,3'-triiodothyronine (T3) (EC50 5x10-1oM), and this effect is blocked by cycloheximide. Chronic treatment of rats with T3 increases pyroglutamyl peptidase I in a number of brain regions and in the pituitary. The induction of pyroglutamyl peptidase I by T3 may contribute to the negative feedback regulation of T3 levels. Pyroglutamyl peptidase II is present in highest concentrations in brain and in retina. The activity of this enzyme in the Y79 retinoblastoma cell is at least tenfold higher than in eight other clonal lines studied. Phorbol ester produces a dose-dependent decrease in pyroglutamyl peptidase II in the Y79 cell. Acute treatment of rats with T3 selectively increases pyroglutamyl peptidase II in frontal cortex and in pituitary. Upon chronic T3 treatment, activity is significantly elevated in frontal cortex and in serum. These results demonstrate that neuropeptide degrading enzymes are subject to regulation by multiple mechanisms and that these enzymes may play a role in the control of the biological activity of neuropeptides. |