We have isolated the TRH receptor (TRH-R) from a rat anterior pituitary cDNA library, determined its sequence and examined its functional characteristics. Expression studies were carried out in Xenopus oocytes and in COS-7 cells. Microinjection of sense RNA transcripts into Xenopus oocytes showed electrophysiological responses of between 800 and 1000 nA under voltage-clamp conditions. COS-7 cells were transiently transfected with the cDNA clone under the control of a cytomegalovirus promoter and inositol phosphate (IP) measurements carried out. In TRH-R transfected cells, TRH (100 nM) produced an approximately twofold increase in total IP production. In-situ hybridization in the rat anterior pituitary revealed a heterogeneous distribution of label, a characteristic pattern of TRH-R expression. The rat 3.3 kb insert coded for a protein of 411 amino acids compared with 393 for the mouse protein. Over its length, the rat TRH-R protein showed considerable homology with that of the mouse, except for a deletion of 232 bp in the 3'-coding region. This deletion did not appear to affect the functional characteristics of the receptor, as shown by electrophysiological studies with Xenopus oocytes and by transfection of the cDNA into COS-7 cells. The sequence given for the 3'-untranslated region is 1.5 kb longer than that reported for the mouse receptor, and extends to the poly(A) tail.
The gonadotrophin-releasing hormone (GnRH) receptor is unlike other G-protein coupled receptors in that the highly conserved amino acids, Asp in the second transmembrane region and Asn in the seventh, are interchanged. Site-directed mutagenesis studies mutated these residues back to their normally conserved positions. Two single mutants Asn87Asp & Asp318Asn and one double mutant Asn87Asp Asp318Asn were transiently expressed in COS-1 cells and their effect on binding to GnRH and inositol phosphate production measured. The single mutant Asp318Asn had no effect on ligand binding but abolished GnRH-dependent inositol phosphate production, whereas mutations Asn87Asp and Asn87Asp Asp318Asn show a complete loss of GnRH binding and subsequent inactivation of its second messenger system.
The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)16.d(AG)21) in the 5' untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3' UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA. The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3' UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3' UTR, allowing transcription to continue into the 3' UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)
In order to facilitate the understanding of gonadotrophinreleasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly 10 , ]-GnRH binding, with a K d value similar to those of both the rat and human forms, but with a greatly reduced B max value. The ED 50 for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser 140 Phe rat GnRH-R mutant was generated. The mutant had a K d value similar to that of the wild-type rat receptor, although the B max value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED 50 value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser 140 Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.
Using a novel technique that facilitates temporal control over the total body force on a liquid, an unexpected scaling relationship was discovered for the collapse time of a liquid bridge. A paramagnetic liquid was suspended between the tips of two collinear rods in a strong magnetic field gradient that was adjusted to compensate gravity. A sudden change of the magnet current, corresponding to a change of Bond number, resulted in a deformation and ultimate collapse of the liquid bridge. The collapse time was found to be independent of the bridge length when other parameters were held constant.
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