FMRFamide‐like peptides encoded on the flp‐18 precursor gene activate two isoforms of the orphan Caenorhabditis elegans G‐protein‐coupled receptor Y58G8A.4 heterologously expressed in mammalian cells

Two alternatively spliced variants of an orphan Caenorhabditis elegans G‐protein‐coupled receptors (GPCRs; Y58G8A.4a and Y58G8A.4b) were cloned and functionally expressed in Chinese hamster ovary (CHO) cells. The Y58G8A.4a and Y58G8A.4b proteins (397 and 433 amino acid residues, respectively) differ both in amino acid sequence and length of the C‐terminal tail of the receptor. A calcium mobilization assay was used as a read‐out for receptor function. Both receptors were activated, with nanomolar potencies, by putative peptides encoded by the flp‐18 precursor gene, leading to their designation as FLP‐18R1a (Y58G8A.4a) and FLP‐18R1b (Y58G8A.4b). Three Ascaris suum neuropeptides AF3, AF4, and AF20 all sharing the same FLP‐18 C‐terminal signature, ‐PGVLRF‐NH 2 , were also potent agonists. In contrast to other previously reported C. elegans GPCRs expressed in mammalian cells, both FLP‐18R1 variants were fully functional at 37°C. However, a 37 to 28°C temperature shift improved their activity, an effect that was more pronounced for FLP‐18R1a. Despite differences in the C‐terminus, the region implicated in distinct G‐protein recognition for many other GPCRs, the same signaling pathways were observed for both Y58G8A.4 isoforms expressed in CHO cells. Gq protein coupling seems to be the main but not the exclusive signaling pathway, because pretreatment of cells with U‐73122, a phospholipase inhibitor, attenuated but did not completely abolish the Ca 2+ signal. A weak Gs‐mediated receptor activation was also detected as reflected in an agonist‐triggered concentration‐dependent cAMP increase. The matching of the FLP‐18 peptides with their receptor(s) allows for the evaluation of the pharmacology of this system in the worm in vivo. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 339–348, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93518/1/20850_ftp.pd

Efficient Interaction of HIV-1 with Purified Dendritic Cells via Multiple Chemokine Coreceptors

HIV-1 actively replicates in dendritic cell (DC)-T cell cocultures, but it has been difficult to demonstrate substantial infection of purified mature DCs. We now find that HIV-1 begins reverse transcription much more efficiently in DCs than T cells, even though T cells have higher levels of CD4 and gp120 binding. DCs isolated from skin or from blood precursors behave similarly. Several M-tropic strains and the T-tropic strain IIIB enter DCs efficiently, as assessed by the progressive formation of the early products of reverse transcription after a 90-min virus pulse at 37°C. However, few late gag-containing sequences are detected, so that active viral replication does not occur. The formation of these early transcripts seems to follow entry of HIV-1, rather than binding of virions that contain viral DNA. Early transcripts are scarce if DCs are exposed to virus on ice for 4 h, or for 90 min at 37°C, conditions which allow virus binding. Also the early transcripts once formed are insensitive to trypsin. The entry of a M-tropic isolates is blocked by the chemokine RANTES, and the entry of IIIB by SDF-1. RANTES interacts with CCR5 and SDF-1 with CXCR4 receptors. Entry of M-tropic but not T-tropic virus is ablated in DCs from individuals who lack a functional CCR5 receptor. DCs express more CCR5 and CXCR4 mRNA than T cells. Therefore, while HIV-1 does not replicate efficiently in mature DCs, viral entry can be active and can be blocked by chemokines that act on known receptors for M- and T-tropic virus

Studies on Solid-Phase Peptide Synthesis: The Synthesis of Glucagon

The feasibility of the stepwise-solid phase approach was studied in the synthesis of the peptide hormone glucagon. The choice of this molecule was based upon: (1) the unusual chemical structure; (2) biological significance

Alignment of the amino acid sequence of zfGLP-1 with sequences of hGLP-1, zebrafish glucagon, human glucagon, exendin-4, exendin(9–39), zfGLP-2 and zebrafish PACAP-38.

<p>Identical amino acids are shown in red. Numbering of hGLP-1 starts at 1 with the amino terminal histidine, corresponding to the biologically active hGLP-1(7–37) and hGLP-1(7–36)amide, to be consistent with histidine 1 in zfGLP-1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167718#sec002" target="_blank">Materials and Methods</a>).</p

ZfGLP-1 (n = 9), hGLP-1 (n = 5), zebrafish glucagon (n = 5), human glucagon (n = 5) and exendin-4 (n = 3) stimulate intracellular cAMP through the recombinant zfGPCR in a similar dose-dependent manner.

<p>Exendin(9–39) (n = 4) and zebrafish PACAP-38 (n = 2) have no effect, and zfGLP-2 (n = 6) has stimulatory effects only at much higher concentrations than the other tested peptides that stimulated cAMP. (n) represents number of separate rounds of transfections. Each data point in the dose-response curve obtained in a single transfection is an average of three separate measurements (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167718#sec002" target="_blank">Materials and Methods</a>). To highlight differences between stimulatory effects of zfGLP-1, zebrafish glucagon, human GLP-1, human glucagon and exendin-4 from the stimulatory effect of zfGLP-2 error bars are shown only for zfGLP-1, zfGLP-2, exendin(9–39) and zfPACAP-38.</p