Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by G q and G i ␣ subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. We report here that an N-terminal domain of RGS4 discriminated among receptor signaling complexes coupled via G q . Accordingly, deletion of the N-terminal domain of RGS4 eliminated receptor selectivity and reduced potency by 10 4 -fold. Receptor selectivity and potency of inhibition were partially restored when the RGS4 box was added together with an N-terminal peptide. In vitro reconstitution experiments also indicated that sequences flanking the RGS4 box were essential for high potency GAP activity. Thus, RGS4 regulates G q class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by G␣ q and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled G q signaling.Heterotrimeric G proteins of the G q class are mediators of Ca 2ϩ responses in animal cells. Signaling is initiated by agonist binding to heptahelical transmembrane receptors complexed with G q ␣␤␥ and phospholipase C-␤ (PLC␤) 1 (1), which generates IP 3 to trigger Ca 2ϩ release from internal stores (2).Many cells express several G q -coupled receptors that regulate the location, intensity, and propagation of intracellular Ca 2ϩ waves. For example, pancreatic acini respond to acetylcholine, bombesin, and cholecystokinin by activating the same set of G q class proteins and mobilizing the same Ca 2ϩ pool, but each receptor evokes distinct patterns of Ca 2ϩ waves (3). Ca 2ϩ release may be regulated by intracellular proteins that interact with guanine nucleotide binding proteins, such as regulators of G protein signaling (RGS) proteins.2 RGS proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by G q and G i ␣ subunits, thus attenuating signaling (5-8). Mammals express over 20 different RGS proteins, of which RGS4 has received the most extensive biochemical characterization (5, 7-12). RGS4 is composed of a central domain of 120 amino acids that is homologous to other RGS proteins, termed the RGS box, flanked by less well conserved N-and C-terminal sequences (13). In rat pancreatic acinar cells, RGS4 preferentially inhibited G q/11 -mediated signaling evoked by carbachol relative to bombesin and cholecystokinin regardless of the identity of the G q class ␣ subunit. 2Regulatory specificity was apparently conferred by direct or indirect interaction between RGS4 and the receptor.In the present study, we used deletion mutations to identify two domains in RGS4 that regulate agonist-dependent Ca 2ϩ signaling. The RGS box accelerates GTP hydrolysis by G␣ q whereas the N terminus conveys high affinity and receptorselective inhibition. These combined activities are required for receptor selectivity and high potency i...

The characteristics of eukaryotic ribosomal proteins PO, P1, and P2 (P proteins) and their antigenic determinants were studied using the sera of patients with systemic lupus erythematosus (SLE). PO, P1, and P2 were isolated as a macromolecular complex by preparative isoelectric focusing and anion-exchange chromatography in the-presence of 6 M urea. The apparent molecular size of the complex was 140 kDa as determined by gel fitration on a Sephadex G-200 column. PO may, therefore, be the eukaryotic equivalent ofEscherichia coli ribosomal protein L10. In addition, all three P proteins were detected in the postribosomal supernatant of HeLa cells, and PO and P1 were found to be more acidic than their ribosome-bound counterparts. Partial proteolysis experiments revealed that SLE anti-P sera recognized one or both ends of the P2 equivalent protein from Artemia saUna (eL12). Sixteen SLE sera containing antibodies to PO, P1, and P2 reacted with a carboxyl-terminal peptide 22 amino acids in length of eL12 and not with an amino-terminal peptide of 20 anmno acids. Even though the carboxyl-terminal peptide completely inhibited the ability of the antiserum to react with all three proteins on an immunological blot, the same peptide produced only small decreases in binding of the SLE antibody to the native, nondenatured P proteins. These findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).Systemic lupus erythematosus (SLE) is an autoimmune disease generally characterized by serum antibodies directed against nuclear proteins and nucleic acids (1). That some patients' sera contain antibodies against ribosomal constituents (2, 3) has long been known, but the identity of these ribosomal antigens has only recently been determined (4). SLE anti-ribosome antibodies show almost exclusive reactivity against three 60S ribosomal subunit phosphoproteins called PO, P1, and P2 (4, 5). These same three proteins are also recognized by a mouse monoclonal antibody raised against chicken ribosomes (6). These "P" proteins have molecular sizes of -38, 19, and 17 kDa, respectively. P1 and P2 are believed to be the eukaryotic equivalent of the Escherichia coli ribosomal protein L12 and have been shown to contain sequences that are highly conserved among eukaryotes (6). Thus, P2 (7) from rat liver shows a high degree of amino acid sequence homology with Artemia salina ribosomal protein eL12 (8) and yeast ribosomal protein YPA1 (9). P1 and P2 also appear to be the functional counterparts of Artemia ribosomal proteins eL12' and eL12 and yeast ribosomal proteins YPA1/YPA2 (10-12). In order to further evaluate the remarkable specificity of the SLE anti-PO, -P1, and -P2 (anti-P) antibodies, we have mapped the antigenic determinant on the P proteins. This determinant is present on all three proteins and is contained within a common sequence of 22 amino acids at the carboxyl terminus of the Artemia r...

A dodecapeptide corresponding to the carboxyl terminus of the lac carrier of Escherichia coli was synthesized, coupled to thyroglobulin, and the conjugate was used to generate site-directed polyclonal antibodies. The antibodies react with the carboxyl-terminal peptide and with the lac carrier protein, while monoclonal antibody 4B1 reacts with intact lac carrier protein, but not with the carboxyl-terminal peptide. Antibody 4B1 binds preferentially to right-side-out membrane vesicles relative to inside-out vesicles, confirming the presence of the 4B1 epitope on the periplasmic surface of the membrane. Alternatively, anti-carboxyl-terminal antibody binds preferentially to inside-out vesicles, demonstrating that the carboxyl terminus of the lac carrier protein is on the cytoplasmic surface. Surprisingly, both antibodies bind to proteoliposomes reconstituted with purified lac carrier protein, and quantitative binding assays indicate that the epitopes are equally accessible. When proteoliposomes containing purified lac carrier protein are digested with carboxypeptidases A and B, binding of anti-carboxyl-terminal antibodies decreases by >80%, while binding of antibody 4B1 and various transport activities remain essentially unchanged. It is suggested that during reconstitution, the lac carrier protein undergoes intramolecular dislocation of the carboxyl terminus with no significant effect on its catalytic activity.The lac carrier protein (i.e., lac permease) in Escherichia coli is an intrinsic membrane protein, the product of the lacY gene, that catalyzes the coupled translocation of 3-galactosides with hydrogen ion in a symport reaction (cf. ref. 1 for a recent review). As such, this protein is representative of a large class of substrate-specific polypeptides that couple downhill movement of a cation to uphill transport of solute in response to a transmembrane electrochemical ion gradient. lac permease has been purified to homogeneity in a functional state, and proteoliposomes reconstituted with a single polypeptide species catalyze all of the transport activities observed in intact cells and right-side-out (RSO) membrane vesicles with full efficiency (1-7). In addition, other similarities between native and purified lac carrier protein have been documented (6, 7), and it is evident from the findings as a whole that /3galactoside transport in E. coli requires a single gene product, that of the lacY gene.The permease is a 46.5-kDa polypeptide containing 417 amino acid residues of known sequence (8). Based on circular dichroic measurements indicating that the protein has an exceptionally high helical content and based on analysis of the sequential hydropathic character of the protein, a secondary structure model has been proposed (9). The model suggests that the protein consists of 12 or 13 hydrophobic ahelical segments that traverse the membrane in a zigzag fashion connected by more hydrophilic loops. Accordingly, the model makes explicit predictions regarding those portions of the molecule that should be acces...

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By comparing the HPLC elution profiles of peptides isolated from different brain regions, two cerebellumspecific species have been identified. The peptides were isolated by sequential chromatography on reverse phase, followed by ion-pairing HPLC using alkane sulfates as pairing reagents. The sequences of both peptides have been determined by gasphase Edman degradation and carboxypeptidase Y analysis and were confirmed by synthesis. The larger peptide, termed cerebellin, is a hexadecamer of primary amino acid sequence NH2 -Ser -Gly -Ser -Ala -Lys -Val -Ala -Phe -Ser-Ala -Ile -ArgSer-Thr-Asn-His-OH. The second peptide is a pentadecamer with a primary sequence identical to residues 2-16 of cerebellin, the nomenclature of which is des-Serl-cerebellin. Subcellular fractionation of cerebellum followed by ion-pairing HPLC analysis shows that both cerebellin and des-Serl-cerebellin are enriched between 2.5-and 4-fold in the P2 crude synaptosomal fraction, suggesting their sequestration in some subcellular particle in vivo.A major goal of developmental neurobiology has been to define the cellular, molecular, and genetic mechanisms that underlie the orderly assembly and maturation of the mammalian nervous system. One fruitful approach to the study of neurodevelopment has been to identify cell-specific markers, usually by immunological techniques, and to assess the expression of these substances in model neural systems (1)(2)(3)(4)(5). However, the general procedure of detecting and monitoring marker expression with antibodies does have a number of limitations. First, only antigenic molecules may be studied. Second, immunocytochemical data are often difficult to quantitate. Finally, subtle modifications of the antigenic determinants, such as glycosylation, may result in ambiguous interpretation of data.In an attempt to circumvent some of the above difficulties, we have tried to characterize brain regions by their native peptide composition. Since the cerebellum has been pointed out to be a particularly useful neural structure for the investigation of the forces regulating neuronal development (6), we have first applied this approach to the rat cerebellum. We report here the isolation and complete primary structures of two related cerebellum-specific peptides that will be shown in further publications to be significant markers for neuronal development in the mammalian cerebellum.MATERIALS AND METHODS Preparation of Tissue Samples for HPLC Analysis. Six-to 8-week-old CD, Sprague-Dawley-derived, rats (Charles River Breeding Laboratories) were decapitated, and their brains were rapidly removed, dissected, and immediately frozen in liquid nitrogen. Prior to HPLC analysis, frozen tissues were homogenized in 10 vol of 6 M guanidine hydrochloride using a Polytron (Brinkmann model PT205T probe) at maximum setting for 30 sec at 4TC. The homogenate was diluted with an equal volume of 0.2 M pyridine/1 M acetic acid and then centrifuged at 25,000 x g for 30 min. The sample was desalted by passing 20 ml of the supernatant over a...