The relaxin peptide family and their novel G-protein coupled receptors

Relaxin-1 is a heterodimeric peptide hormone primarily produced by the pregnant corpus luteum and/or placenta and is involved in many essential physiological processes centered on its action as a potent extracellular matrix (ECM) remodeling agent. Insulin-like peptide 3 (INSL3), also known as relaxin-like factor, is predominantly expressed in the Leydig cells of the testes and is an important mediator of testicular descent. The relaxin-1 equivalent peptide in humans is actually the product of the human RLN2 gene, human 2 (H2) relaxin. Recently identified and thought to be the ancestral relaxin, relaxin-3 is specifically expressed in the nucleus incertus of the mouse and rat brain and is most likely an important neuropeptide. Each of the hormones above act on cell membrane G-protein coupled receptors (GPCRs). The relaxin-1 receptor is leucine-rich repeat-containing GPCR 7 (LGR7) whereas INSL3 acts on the closely related LGR8. These receptors have large extra-cellular domains containing multiple leucine-rich repeats (LRRs) and a unique LDL receptor-like cysteine-rich motif (LDLR-domain). Relaxin-3 will bind and activate LGR7 with 50-fold lower activity than H2 relaxin. Two relaxin-3 selective GPCRs; somatostatin and angiotensin like peptide receptor (SALPR) and GPCR 142 were recently identified, these type I GPCRs are unrelated to LGR7 and LGR8. The discovery and characterisation of these receptors is greatly aiding the quest to unravel the mechanics of these important hormones, however with three other family members, insulin-like peptides 4–6 (INSL4, INSL5 and INSL6) with unknown functions and unidentified receptors, there is still much to be learnt about this hormone family.     

Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR

We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10⁴ cells are required for the analysis.     

Hypothesis for a serine proteinase-like domain at the COOH terminus of Slowpoke calcium-activated potassium channels

Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein with three disulfide bonds that belongs to the Kunitz family of serine proteinase inhibitors. BPTI is an extremely potent inhibitor of trypsin, but it also specifically binds to various active and inactive serine proteinase homologs with KD values that range over eight orders of magnitude. We previously described an interaction of BPTI at an intracellular site that results in the production of discrete subconductance events in large conductance Ca2+ activated K+ channels (Moss, G.W.J., and E. Moczydlowski. 1996, J. Gen. Physiol, 107:47-68). In this paper, we summarize a variety of accumulated evidence which suggests that BPTI binds to a site on the KCa channel protein that structurally resembles a serine proteinase. One line of evidence includes the finding that the complex of BPTI and trypsin, in which the inhibitory loop of BPTI is masked by interaction with trypsin, is completely ineffective in the production of substate events in the KCa channel. To further investigate this notion, we performed a sequence analysis of the alpha-subunit of cloned slowpoke KCa channels from Drosophila and mammals. This analysis suggests that a region of approximately 250 residues near the COOH terminus of the KCa channel is homologous to members of the serine proteinase family, but is catalytically inactive because of various substitutions of key catalytic residues. The sequence analysis also predicts the location of a Ca(2+)-binding loop that is found in many serine proteinase enzymes. We hypothesize that this COOH-terminal domain of the slowpoke KCa channel adopts the characteristic double-barrel fold of serine proteinases, is involved in Ca(2+)-activation of the channel, and may also bind other intracellular components that regulate KCa channel activity.     

Role of 5'-guanylylimidodiphosphate in the activation of adenylyl cyclase by parathyroid hormone.

We have studied the effects of guanylylimidodiphosphate (Gpp(NH)p), an analogue of GTP, on the stimulation of renal cortical adenylyl cyclase by bovine parathyroid hormone (bPTH, or bPTH-(1-84)). Incubation of canine renal membranes with bPTH-(3-34), a specific antagonist of parathyroid hormone, in either the presence or absence of Gpp(NH)p, prevented subsequently added bPTH-(1-84) from stimulating adenylyl cyclase. The addition of the antagonist to a cyclase system previously activated by both bPTH-(1-84) and Gpp(NH)p, however, produced no inhibition of enzyme activity. Removal of bPTH by washing the membranes virtually abolished activity, but washing after addition of bPTH plus Gpp(NH)p did not prevent continued accumulation of cAMP. The persistence of the activity of the enzyme brought about by the addition of Gpp(NH)p plus bPTH, despite washing or addition of specific inhibitor of bPTH action, indicates that the activity of the hormone-specific adenylyl cyclase in membrane suspensions is independent of cintinuous occupancy of the peptide-hormone receptor by bPTH in the presence of the guanyl-nucleotide analogue.     

Development of the hypothalamic-pituitary-axis in the ovine fetus: ontogeny of action of ovine corticotropin-releasing factor

In samples from twenty chronically cannulated ovine fetuses the plasma immunoreactive adrenocorticotrophin (ACTH) concentrations were 12.5 +/- 3.2(8), 15.2 +/- 4.1(9) and 21.2 +/- 5.6(8) pg/ml at periods, prior to parturition, of -30 to -35, -25 to -29 and -20 to -24 days respectively. Values are mean +/- SEM (number of samples). These values were not significantly different from each other but were significantly lower (P less than 0.02) than values in the next two age groups -36.0 +/- 4.9(7) pg/ml at -19 to -15 days, and 39.6 +/- 6.6(11) pg/ml at -14 to -9 days. A further significant increase (P less than 0.05) occurred in the -8 to -3 day period, ACTH being 53.9 +/- 5.4(12) pg/ml. On day of delivery two samples had values of 325 and 360 pg/ml. A single injection, intravenously of 1.0 microgram ovine corticotrophin-releasing factor (O-CRF), caused a significant increase in fetal plasma ACTH concentrations in fetuses of -6 to -23 days prior to delivery but not in fetuses -24 to -35 days prior to parturition. The maximum values of ACTH after O-CRF were significantly greater in fetuses -2 to 0 days prior to parturition than in younger fetuses (P less than 0.01). In 6 experiments in 4 fetuses (parturition -1 to -13 days) the effect of 1.0 microgram O-CRF persisted for at least 2.5 h. The results support the hypothesis that the pituitary release of ACTH changes sensitivity to hypothalamic O-CRF at least twice during the last fifth of gestation; an increasing sensitivity is seen as term approaches.     

Alpha-inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin

Inhibin is a glycoprotein hormone composed of two nonidentical subunits. It is produced by the ovary and testis and plays a vital role in gonadal function by inhibiting the secretion of FSH. More recently, additional activities associated with inhibin peptides have been identified. Inhibin heterodimers (alpha-beta) are reported to act directly on ovarian granulosa cells and inhibit estrogen production induced by FSH. Furthermore, homodimers of beta-inhibin subunits stimulate the secretion of FSH, an activity that is directly opposite to that of inhibin. Each of these inhibin-related activities are concerned with the hypothalamic-pituitary-gonadal axis. We have investigated further the complexity of inhibin activity by determining whether inhibin genes are expressed in nongonadal tissue. RNA hybridization experiments demonstrate that the alpha-inhibin gene is expressed in the sheep adrenal cortex and hybridization histochemistry shows that this gene is expressed in each of the functional zones within the cortex. Dot blot analysis showed that the level of alpha mRNA within the adrenal is influenced by ACTH, one of the major regulators of adrenal cortex function. These observations imply that there are inhibin-related peptides not directly associated with the gonads. beta-inhibin gene expression was not clearly detected in the adrenal and we conclude that if expression occurs then it does so at extremely low levels.