Elo Eriste

Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

GPCR135, publicly known as somatostatin- and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35S-labeled guanosine 5′-O-(3-thiotriphosphate) (GTPγS) incorporation in cells over-expressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTPγS binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC50 values of 0.25 and 0.35 nM, respectively. 125I-Relaxin-3 binds GPCR135 at high affinity with a Kd value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry.

Evidence for non-isostructural replacement of Zn2+ with Cd2+ in the β-domain of brain-specific metallothionein-3

Metallothionein‐3 (MT‐3) is a brain‐specific MT, which is downregulated in Alzheimers disease. The N‐terminal region of CdMT‐3 is highly dynamic and has escaped structural characterization by nuclear magnetic resonance. We have used electrospray ionization mass spectrometry to probe conformational states of cadmium‐ and zinc‐substituted metalloforms of MT‐3 and can demonstrate that the N‐terminal β‐domain of MT‐3 filled with Cd2+ has a more open conformation than that filled with Zn2+. The results suggest that the larger Cd2+ ions cannot isostructurally replace zinc in the β‐domain of MT‐3 whereas in the case of MT‐1 and MT‐2 the replacement is isostructural. Specific metal binding properties of the β‐domain of MT‐3 may be essential for fulfilling the specific role of MT‐3 in the brain.

Ser13-phosphorylated PYY from porcine intestine with a potent biological activity

We have isolated a posttranslationally modified form of peptide YY (PYY) from porcine intestine and shown by MALDI‐TOF and electrospray tandem mass spectrometry that it is phosphorylated at Ser13. Phospho‐PYY exhibits high affinity for binding to neuropeptide Y (NPY) receptors Y1, Y2 and Y5. The IC50 values with the Y1, Y2, and Y5 receptor subtypes were for NPY 2.4, 3.1, and 3.3 nM, for PYY 2.3, 0.94, and 3.2 nM, and for phospho‐PYY 4.6, 2.2, and 5.5 nM, respectively. Phospho‐PYY potently inhibits forskolin‐stimulated cAMP accumulation in SK‐N‐MC cells with an IC50 value of 0.5 nM compared to 0.15 nM for non‐phosphorylated PYY. The finding of phosphorylation of PYY is unusual among hormonal peptides, and emphasizes the importance of direct protein analysis of gene products.

Purification of recombinant human apometallothionein-3 and reconstitution with zinc.

Metallothioneins (MT) are small cysteine-rich proteins, expressed in many life forms. They are involved primarily in the metabolism of zinc and copper, and in metal detoxification processes. Metallothionein-3 is a mammalian brain-specific MT, which is down-regulated in Alzheimers disease brains. In this report, we describe a new procedure for purification of recombinant human apo-MT-3 by three steps, size exclusion at neutral pH, followed by cation-exchange and reverse-phase HPLC, both at low pH. Purified apo-MT-3 was reconstituted with seven Zn(2+) ions and reconstitution products were analyzed with electrospray ionization mass spectrometry. The mass spectrum of reconstituted ZnMT-3 was identical with that of native ZnMT-3 isolated by size exclusion chromatography proving the efficiency of the reconstitution process. It showed that ZnMT-3 exists in solution as a dynamic mixture of several metalloforms, where the main metalloform is Zn(7)MT-3 and minor forms are Zn(6)MT-3 and Zn(8)MT-3.

A C-terminally elongated form of PHI from porcine intestine

Abstract. A C-terminally elongated form of peptide histidine isoleucine amide (PHI) was isolated from porcine intestine based on its effect on cAMP production in IMR-32 cells. The structure was determined by amino acid sequence analysis of tryptic fragments and by mass spectrometry. The peptide has 42 amino acid residues like those described from human, rat and mouse, but the amino acid sequence of the C-terminal extension of pig PHI is unique. Unlike the other peptides, it has a C-terminal Ala and it differs at five positions from the human form and at six positions from the rat form, while the human and the rat forms differ by only two substitutions. To avoid confusion arising from different C-terminal residues, a unifying nomenclature is proposed: PHI-27 for the hormone and PHI-42 for the elongated product.

Extracellular Acidification: A Novel Detection System for Ligand/Receptor Interactions

Microphysiometry is a technique that measures changes in the metabolic activity of cells (acid secretion) as a response to extracellular stimuli (McConnell et al., 1992). Cellular metabolism in response to the stimulation leads to consumption of glucose, synthesis and use of ATP, and production of several acidic metabolites such as lactic acid and bicarbonate. They are finally excreted from the cell by passive and active transport systems. The resulting change in the metabolic rate leads usually to a measurable increase in acid excretion from the cell, but may in a few cases lead to a decrease. Measurements of extracellular acidification rates (ECAR) can be carried out using an instrument with a silicon pH sensor, Cytosensor, and can trace when cell surface receptors have been stimulated by various signalling substances, such as hormones, neurotransmitters or growth factors.