A G protein gamma subunit shares homology with ras proteins

Guanine nucleotide binding proteins (G proteins) that transduce signals from cell surface receptors to effector molecules are made up of three subunits, alpha, beta, and gamma. A complementary DNA clone that encodes a 71-amino acid protein was isolated from bovine brain; this protein contains peptide sequences that were derived from the purified gamma subunit of Gi and Go. The primary sequence of this G protein gamma subunit (G gamma) has 55 percent homology to the gamma subunit of transducin (T gamma) and also has homology to functional domains of mammalian ras proteins. The probe for isolating the clone was generated with the use of the polymerase chain reaction (PCR). The extent of divergence between T gamma and G gamma, the isolation of homologous PCR-generated fragments, and the differences between the predicted amino acid sequence of G gamma and that derived from the gamma subunit of Gi and Go indicate that gamma subunits are encoded by a family of genes.     

Somatic mutation in genes for the variable portion of the immunoglobulin heavy chain

The size of the gene pool potentially encoding antibodies to p-azophenyl arsonate has been examined. A heavy chain-specific full-length complementary DNA clone has been constructed with the use of messenger RNA from a hybridoma that produces antibodies to the arsonate hapten and bears nearly a full complement of the determinants comprising the cross-reactive idiotype (CRI). The sequences of both the complementary DNA clone and the corresponding immunoglobulin heavy chain have been independently determined. A probe for the variable region gene was prepared from the original heavy chain complementary DNA clone and used to analyze, by Southern filter hybridization, genomic DNA from both A/J (CRI positive) and BALB/c (CRI negative) mice. Approximately 20 to 25 restriction fragments containing "germline" variable region gene segments were detected in both strains, and many are shared by both, Since 35 CRI-positive heavy chains have been partially sequenced thus far and 31 are different, the results of the hybridization analysis suggest that somatic mutation events involving the variable region gene segments of the heavy chain play a role in the origin of the amino acid sequence diversity seen in this system.     

Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease

Four clones were isolated from an adult human brain complementary DNA library with an oligonucleotide probe corresponding to the first 20 amino acids of the beta peptide of brain amyloid from Alzheimer's disease. The open reading frame of the sequenced clone coded for 97 amino acids, including the known amino acid sequence of this polypeptide. The 3.5-kilobase messenger RNA was detected in mammalian brains and human thymus. The gene is highly conserved in evolution and has been mapped to human chromosome 21.     

Sequence and structure of a human glucose transporter

The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic alpha helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.     

Golf: an olfactory neuron specific-G protein involved in odorant signal transduction

Biochemical and electrophysiological studies suggest that odorants induce responses in olfactory sensory neurons via an adenylate cyclase cascade mediated by a G protein. An olfactory-specific guanosine triphosphate (GTP)-binding protein alpha subunit has now been characterized and evidence is presented suggesting that this G protein, termed Golf, mediates olfaction. Messenger RNA that encodes Golf alpha is expressed in olfactory neuroephithelium but not in six other tissues tested. Moreover, within the olfactory epithelium, Golf alpha appears to be expressed only by the sensory neurons. Specific antisera were used to localize Golf alpha protein to the sensory apparatus of the receptor neurons. Golf alpha shares extensive amino acid identity (88 percent) with the stimulatory G protein, Gs alpha. The expression of Golf alpha in S49 cyc- kin- cells, a line deficient in endogenous stimulatory G proteins, demonstrates its capacity to stimulate adenylate cyclase in a heterologous system.     

Identification of specific transducin alpha subunits in retinal rod and cone photoreceptors

Transducin is a guanyl nucleotide-binding protein that couples rhodopsin photolysis to hydrolysis of guanosine 3',5'-monophosphate in rod photoreceptor cells of vertebrate retinas. Several complementary DNA clones encoding transducin subunits have recently been characterized. One clone, isolated from a bovine retina complementary DNA library, encodes a previously unidentified polypeptide with an amino acid sequence 78% identical to the sequence of the alpha subunit of bovine rod outer segment transducin. Antibodies to a synthetic peptide with amino acid sequence derived specifically from this novel polypeptide recognize a 41-kilodalton polypeptide in homogenates of bovine retina. Localization of this polypeptide in bovine retina by indirect immunofluorescence demonstrates that it is expressed only in cone outer segments. Antibodies to specific sequences found only in the rod transducin alpha subunit recognize a polypeptide localized only in the rod outer segment. Therefore, bovine rod and cone cells each express structurally related yet significantly different forms of transducin.     

Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor

Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.     

Carboxyl terminal domain of Gs alpha specifies coupling of receptors to stimulation of adenylyl cyclase

The alpha subunits of Gs and Gi link different sets of hormone receptors to stimulation and inhibition, respectively, of adenylyl cyclase. A chimeric alpha i/alpha s cDNA was constructed that encodes a polypeptide composed of the amino terminal 60% of an alpha i chain and the carboxyl terminal 40% of alpha s. The cDNA was introduced via a retroviral vector into S49 cyc- cells, which lack endogenous alpha s. Although less than half of the hybrid alpha chain is derived from alpha s, its ability to mediate beta-adrenoceptor stimulation of adenylyl cyclase matched that of the normal alpha s polypeptide expressed from the same retroviral vector in cyc- cells. This result indicates that carboxyl terminal amino acid sequences of alpha s contain the structural features that are required for specificity of interactions with the effector enzyme, adenylyl cyclase, as well as with the hormone receptor.     

Sequence of the alpha subunit of photoreceptor G protein: homologies between transducin, ras, and elongation factors

A bovine retinal complementary DNA clone encoding the alpha subunit of transducin (T alpha) was isolated with the use of synthetic oligodeoxynucleotides as probes, and the complete nucleotide sequence of the insert was determined. THe predicted protein sequence of 354 amino acids includes the known sequences of four tryptic peptides and sequences adjacent to the residues that undergo adenosine diphosphate ribosylation by cholera toxin and pertussis toxin. On the basis of homologies to other proteins, such as the elongation factors of protein synthesis and the ras oncogene proteins, regions are identified that are predicted to be acylated and involved in guanine nucleotide binding and hydrolysis. Amino acid sequence similarity between T alpha and ras is confined to these regions of the molecules.     

Sequence and expression of human estrogen receptor complementary DNA

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.     

Cloning of human androgen receptor complementary DNA and localization to the X chromosome

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.     

Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart

Neuronal cells express a pp60c-src variant that displays an altered electrophoretic mobility and a different V8 peptide pattern relative to pp60c-src expressed in tissues of non-neuronal origin. To determine whether the neuronal form of pp60c-src is encoded by a brain-specific messenger RNA, a mouse brain complementary DNA (cDNA) library was screened with a chicken c-src probe and a 3.8-kilobase c-src cDNA clone was isolated. This clone encodes a 60-kilodalton protein that differs from chicken or human pp60c-src primarily in having six extra amino acids (Arg-Lys-Val-Asp-Val-Arg) within the NH2-terminal 16 kilodaltons of the molecule. S1 nuclease protection analysis confirmed that brain c-src RNA contains an 18-nucleotide insertion at the position of the extra six amino acids. This insertion occurs at a position that corresponds to a splice junction in the chicken and human c-src genes. The isolated c-src cDNA clone encodes a protein that displays an identical V8 peptide pattern to that observed in pp60c-src isolated from tissues of neuronal origin.     

Isolation of an olfactory cDNA: similarity to retinol-binding protein suggests a role in olfaction

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.     

Expression of a cloned rat brain potassium channel in Xenopus oocytes

Potassium channels are ubiquitous membrane proteins with essential roles in nervous tissue, but little is known about the relation between their function and their molecular structure. A complementary DNA library was made from rat hippocampus, and a complementary DNA clone (RBK-1) was isolated. The predicted sequence of the 495-amino acid protein is homologous to potassium channel proteins encoded by the Shaker locus of Drosophila and differs by only three amino acids from the expected product of a mouse clone MBK-1. Messenger RNA transcribed from RBK-1 in vitro directed the expression of potassium channels when it was injected into Xenopus oocytes. The potassium current through the expressed channels resembles both the transient (or A) and the delayed rectifier currents reported in mammalian neurons and is sensitive to both 4-aminopyridine and tetraethylammonium.     

Beta-adrenergic receptor kinase: primary structure delineates a multigene family

The beta-adrenergic receptor kinase (beta-ARK), which specifically phosphorylates only the agonist-occupied form of the beta-adrenergic and closely related receptors, appears to be important in mediating rapid agonist-specific (homologous) desensitization. The structure of this enzyme was elucidated by isolating clones from a bovine brain complementary DNA library through the use of oligonucleotide probes derived from partial amino acid sequence. The beta-ARK cDNA codes for a protein of 689 amino acids (79.7 kilodaltons) with a protein kinase catalytic domain that bears greatest sequence similarity to protein kinase C and the cyclic adenosine monophosphate (cyclic AMP)--dependent protein kinase. When this clone was inserted into a mammalian expression vector and transfected into COS-7 cells, a protein that specifically phosphorylated the agonist-occupied form of the beta 2-adrenergic receptor and phosphorylated, much more weakly, the light-bleached form of rhodopsin was expressed. RNA blot analysis revealed a messenger RNA of four kilobases with highest amounts in brain and spleen. Genomic DNA blot analysis also suggests that beta-ARK may be the first sequenced member of a multigene family of receptor kinases.     

Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells

The sequence of the human epidermal growth factor (EGF) receptor shows great homology with the avian erythroblastosis virus v-erb B oncogene, raising the possibility that the receptor gene is identical to the c-erb B protooncogene. Human A431 epidermoid carcinoma cells, which have an unusually high number of EGF receptors, were examined to determine whether elevated EGF receptor levels correlate with gene amplification. Southern blots of genomic DNA's from A431 and other human cell lines were probed with either a v-erb B gene fragment or a human EGF receptor complementary DNA clone (pE7), previously isolated from an A431 complementary DNA library. When either probe was used to analyze Eco RI- or Hind III-generated DNA fragments, EGF receptor DNA sequences were amplified about 30-fold in A431. Differences in the banding pattern of A431 DNA fragments relative to normal fibroblast DNA indicate the occurrence of a rearrangement in the region of the receptor gene. Furthermore, A431 cells contain a characteristic, prominent 2.9-kilobase RNA. These results are consistent with the hypothesis that, in A431 cells, gene amplification, possibly associated with a translocation event, may result in the overproduction of EGF receptor protein or the appearance of the transformed phenotype (or both).     

Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) participates in many processes including early developmental events, angiogenesis, wound healing, and maintenance of neuronal cell viability. A 130-kilodalton protein was isolated on the basis of its ability to specifically bind to bFGF. A complementary DNA clone was isolated with an oligonucleotide probe corresponding to determined amino acid sequences of tryptic peptide fragments of the purified protein. The putative bFGF receptor encoded by this complementary DNA is a transmembrane protein that contains three extracellular immunoglobulin-like domains, an unusual acidic region, and an intracellular tyrosine kinase domain. These domains are arranged in a pattern that is different from that of any growth factor receptor described.     

cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily

Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.     

A G protein mutant that inhibits thrombin and purinergic receptor activation of phospholipase A2

The stimulation of phospholipase A2 by thrombin and type 2 (P2)-purinergic receptor agonists in Chinese hamster ovary cells is mediated by the G protein Gi. To delineate alpha chain regulatory regions responsible for control of phospholipase A2, chimeric cDNAs were constructed in which different lengths of the alpha subunit of Gs (alpha s) were replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). When a carboxyl-terminal chimera alpha s-i(38), which has the last 38 amino acids of alpha s substituted with the last 36 residues of alpha i2, was expressed in Chinese hamster ovary cells, the receptor-stimulated phospholipase A2 activity was inhibited, although the chimera could still activate adenylyl cyclase. Thus, alpha s-i(38) is an active alpha s, but also a dominant negative alpha i molecule, indicating that the last 36 amino acids of alpha i2 are a critical domain for G protein regulation of phospholipase A2 activity.