Structural organization and chromosomal localization of three human galanin receptor genes.

Human galanin receptor subtypes GALR1, GALR2, and GALR3 are encoded by separate genes that are located on human chromosomes 18q23, 17q25.3, and 22q13.1, respectively. The exon:intron organization of the gene encoding GALR2 (GALNR2) and GALR3 (GALNR3) is conserved, with exon 1 encoding the NH2-terminus to the end of transmembrane domain 3 and exon 2 encoding the remainder of the receptor, from the second intracellular loop to the COOH-terminus. This conservation of structural organization is indicative of a common evolutionary origin for GALNR2 and GALNR3. The exon:intron organization of the gene encoding GALR1 (GALNR1) is different from that of GALNR2 and GALNR3, with exon 1 encoding the NH2-terminus to the end of transmembrane domain 5, exon 2 encoding the third intracellular loop, and exon 3 encoding the remainder of the receptor, from transmembrane domain 6 to the COOH-terminus. The structural organization of GALNR1 suggests convergent evolution for this gene and represents a structural organization that is unique among genes encoding G-protein-coupled receptors.

Structural organization of the mouse and human GALR1 galanin receptor genes (Galnr and GALNR) and chromosomal localization of the mouse gene.

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15-20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouse Galnr gene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the human GALNR gene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.

Intron 17 of the human retinoblastoma susceptibility gene encodes an actively transcribed G protein-coupled receptor gene.

The human retinoblastoma susceptibility gene, a member of the tumor suppressor gene family, is located on chromosome 13q14.12-13q14.2 and consists of 27 exons that are distributed over 180 kb. This study shows that intron 17, the largest in size, consisting of nearly 72,000 bp, contains an open reading frame encoding a novel G protein-coupled receptor in the reverse orientation relative to the transcription of the retino-blastoma susceptibility gene. Correction of a frameshift mutation revealed that this novel G protein-coupled receptor is the human homolog of a chicken T-cell-specific receptor cDNA. This is an additional description of an actively transcribed protein-encoding gene positioned within an intron of another gene, suggesting that introns can have important structural functions.

Molecular cloning, characterization, and localization of the human homolog to the reported bovine NPY Y3 receptor: lack of NPY binding and activation.

A cDNA clone encoding the human homolog of the bovine cDNA clone LCR1 was isolated from a human lung cDNA library. The 1,670-bp-long nucleotide sequence predicts a single open reading frame of 352 amino acids, with a 92% amino acid identity to a bovine sequence reported to represent the neuropeptide Y (NPY) Y3 receptor. The amino acid sequence shares features common to many other G-protein-coupled receptors, including the seven transmembrane regions and putative glycosylation and phosphorylation sites. Polymerase chain reaction (PCR) analysis of human-hamster hybrid cell DNA reveals that the corresponding gene is located on human chromosome 2. Although the ligand for the bovine receptor has previously been identified as NPY in binding studies, extensive analysis with the human homolog transfected in several different cell lines failed to confirm this classification. Furthermore, the receptor shows 36% identity to both the human interleukin-8 (IL-8) and angiotensin II receptors but only 21% identity to the human NPY Y1 receptor. In addition, NPY and a number of other ligands fail to induce any change in cytosolic calcium levels in transfected cells, suggesting that this clone represents a novel neuropeptide receptor.

Isoelectric charge of recombinant human follicle-stimulating hormone isoforms determines receptor affinity and in vitro bioactivity.

Recombinant human FSH (rhFSH) was obtained by expressing the human FSH alpha- and beta-subunit complementary DNAs in the chinese hamster ovary cell line. Isoforms of rhFSH were resolved into specific isoelectric (pI) fractions by chromatofocusing. rhFSH isoforms ranged from pI 3.0-5.5 with a modal value of pI 4.2. Analysis of the biological activity of specific pI isoforms of rhFSH was undertaken using both the rat granulosa cell aromatase (in vitro) bioassay and a RRA. More acidic isoforms (e.g. pI 3.5) showed significantly lower affinity (P < 0.05) for rat testicular FSH receptors than did the less acidic isoforms (e.g. pI 4.8). Consistent with the receptor binding affinity data, the more acidic fractions resulted in significantly less activation (P < 0.05) of rat granulosa cell aromatase activity, as measured by estrogen production, than did the less acidic isoforms. The observed bioactivities and their correlation with the pI values of the rhFSH isoforms are consistent with observations of differing bioactivities seen in both pituitary and urinary FSH isoforms. These results demonstrate that rhFSH, made in the chinese hamster ovary cell line, is both biologically active and has isoform profiles, and presumably carbohydrate structures, that closely resemble those seen in natural hFSH.

Cloned human neuropeptide Y receptor couples to two different second messenger systems.

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiological activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system. Two major subtypes of NPY receptor (Y1 and Y2) have been defined by pharmacological criteria. We report here the molecular cloning of a cDNA sequence encoding a human NPY receptor and the corrected sequence for a rat homologue. Analysis of this sequence confirms that the receptor is a member of the G protein-coupled receptor superfamily. When expressed in Chinese hamster ovary (CHO) or human embryonic kidney (293) cells, the receptor exhibits the characteristic ligand specificity of a Y1 type of NPY receptor. In the 293 cell line, the receptor is coupled to a pertussis toxin-sensitive G protein that mediates the inhibition of cyclic AMP accumulation. In the CHO cell line, the receptor is coupled not to the inhibition of adenylate cyclase but rather to the elevation of intracellular calcium. These results demonstrate that second messenger coupling of the NPY-Y1 receptor is cell type specific, depending on the specific repertoire of G proteins and effector systems present in any cell type.

Analysis of cDNA clones encoding the entire B-26 region of human apolipoprotein B.

We report the characterization of intestine and liver cDNA clones for human apolipoprotein B (apoB) that map to the 5' end of the mRNA. The protein sequence encoded by the 5011 nucleotides derived from sequence analysis of these clones includes 1643 amino acid residues of the mature protein of Mr 184,000. The amino acid sequence at the amino terminus of B-74 peptide was determined and mapped to residue 1298. The size (Mr 145,700) and amino acid composition of the B-26 region encoded by these clones (including amino acid residues 1-1297) closely match the values obtained from the B-26 peptide. The amino acid sequence of peptide B-100 at the junction of peptides B-26 and B-74 (Phe-Lys decreases- Ser) shows structural homology to the site on human kininogen (Phe-Arg decreases- Ser) that is cleaved by the protease plasma kallikrein. The encoded protein contains five potential N-glycosylation sites and several regions in which the hydroxyamino acids, serine and threonine, are present in high abundance. The protein sequence presented in this report represents approximately 30% of the total B-100 protein and will aid in the characterization of additional cDNA clones.

Primary structure of the human renin gene.

The gene encoding human renin has been isolated on two overlapping clones from a bacteriophage lambda library of human DNA. The entire gene spans about 12,000 bp and contains 10 exons separated by 9 intervening sequences. The gene structure is similar to that of human pepsinogen in terms of overall size, homology in the coding regions, position of introns, and sizes of the exons, suggesting that the two genes are evolutionarily related. However, a novel exon coding for only three amino acids was detected that is not present in the pepsinogen gene and whose amino acids are also not found in mouse renin. Although the nucleotide sequence of the 5'-flanking DNA differs from that of the pepsinogen gene, in both cases this region contains a structure of almost perfect dyad symmetry which immediately precedes the TATA box and may have functional importance. Furthermore, sequences resembling the putative consensus sequence for glucocorticoid regulation of gene expression are located approximately 200 and 300 bp upstream from the gene. The overall structural anatomy suggests that the human renin gene evolved by mechanisms that include a duplication of exon segments, particularly those containing the codons for the catalytically important aspartate residues, together with the insertion of other exon and flanking DNA structures. An analysis of human chromosomal DNA demonstrates that there is only one gene with high homology to human renin.