The cholecystokinin-A receptor mediates inhibition of food intake yet is not essential for the maintenance of body weight

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility.Tyrosine O-sulfation is a widespread post-translational modification that was first described about 50 years ago (1, 2). Tyrosine-sulfated proteins and/or tyrosylprotein sulfotransferase (TPST) 5 activity have been described in many species throughout the plant and animal kingdoms, including Volvox carteri, one of the earliest multicellular organisms. At this time, 37 tyrosine-sulfated proteins have been identified in humans, many of which play important roles in inflammation, hemostasis, immunity, and other processes (3-5). These include certain adhesion molecules, G-protein-coupled receptors, coagulation factors, serpins, extracellular matrix proteins, hormones, and others. It has been demonstrated that some of these proteins require tyrosine sulfation for optimal function (3). Nevertheless, it is very likely that we are only beginning to appreciate the complexity of the TPST substrate repertoire.In mice and humans, tyrosine O-sulfation is mediated by one of two tyrosylprotein sulfotransferases, called TPST-1 and TPST-2, which are localized to the trans-Golgi network (7-9). Mouse TPST-1 and -2 are 370-and 376-residue type II transmembrane proteins, respectively. Each has a short N-terminal cytoplasmic domain, followed by a single ϳ17-residue transmembrane domain, a membrane-proximal ϳ40-residue stem region, and a luminal catalytic domain containing four conserved cysteine residues and two N-glycosylation sites. The amino acid sequence of human and mouse TPST-1 are ϳ96% identical, and human and mouse TPST-2 have a similar degree of identity. TPST-1 is ϳ65-67% identical to TPST-2 in both humans and mice. Both isoenzymes are broadly expressed in human and murine tissues and cell lines and are co-expressed in most, if not all, cell types (3). However, the relative abundance of the two proteins in tissues and cells is uncertain. The human TPST1 and TPST2 genes are on 7q11.21 and 22q12.1, respectively. The mouse Tpst1 and Tpst2 genes are ϳ18.5 megabase pairs apart on chromosome 5.In vitro studies using synthetic peptide acceptors indicate that the two TPST isoenzymes differ in substrate preference. Peptides modeled on the N terminus of P-selectin glycoprotein ligand-1 are sulfated by the two isoenzymes with equal efficiency. In contrast, pept...

Section: Discussionmentioning

confidence: 99%

Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme That Catalyzes Post-translational Protein Tyrosine O-Sulfation, Causes Male Infertility

Borghei

Ouyang

Westmuckett

et al. 2006

“…Labeled peptides were prepared and purified as described previously (22). CCKAR Ϫ/Ϫ mice were generated as described previously (23). Collagenase (grade) was purchased from Worthington Biochemicals (Freehold, NJ).…”

Section: Methodsmentioning

confidence: 99%

Molecular Mechanism Underlying Partial and Full Agonism Mediated by the Human Cholecystokinin-1 Receptor

Archer-Lahlou

Escrieut

Clerc

et al. 2005

The cholecystokinin-1 receptor (CCK1R) is a G protein-coupled receptor (GPCR) that regulates important physiological functions. As for other GPCRs, the molecular basis of full and partial agonism is still far from clearly understood. In the present report, using both laboratory experiments and molecular modeling approaches, we have investigated the partial agonism mechanism of JMV 180, on the human CCK1R. We first showed that efficacy of the CCK1R to activate phospholipase C is dependent on the correct orientation of the C-terminal end of peptidic ligands toward residue Phe 330 of helix VI. We have previously reported that a single mutation of Met 121 (helix III) markedly reduced the receptor-mediated inositol phosphate production upon stimulation by CCK. Computational simulations predicted that residue 121 affected orientation of the C-terminal end of CCK, thus suggesting that the molecular complex with a reduced inositol phosphate production observed with the mutated CCK1R resembles that resulting from binding of JMV 180 to the WT-CCK1R. Pharmacological, biochemical, and functional characterizations of the two receptor⅐ligand complexes with decreased abilities to signal were carried out in different cell types. We found that they presented the same features, such as total dependence of inositol phosphate production to G␣ q expression, single affinity of binding sites, insensitivity of binding to non-hydrolyzable GTP, absence of GTP␥ [S 35 ] binding following agonist stimulation, similarity of dose-response curves for amylase secretion, and incapacity to induce acute pancreatitis in pancreatic acini. We concluded that helices VI and III of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the C-terminal ends of peptidic agonists toward Phe 330 of helix VI is responsible for extent of phospholipase C activation through G␣ q coupling. Given the potential therapeutic interest of partial agonists such as JMV 180, our structural data will serve for target structure-based design of new CCK1R ligands.

“…CCK A R Ϫ/Ϫ mice were generated as described previously (11). 129/SVIMJ control mice were purchased from The Jackson Laboratory (Bar Harbor, Maine).…”

Section: Methodsmentioning

confidence: 99%

Species Differences between Rat and Mouse CCKAReceptors Determine the Divergent Acinar Cell Response to the Cholecystokinin Analog JMV-180

Ji¹,

Kopin²,

Logsdon³

2000

Self Cite

The cholecystokinin (CCK) analog JMV-180 acts as a partial agonist in rats and a full agonist in mice. Whether this functional variability is due to species differences in CCK receptor structure or to alterations in the cellular environment is unknown. To address this question, an adenoviral construct encoding the rat CCK A receptor (AdCCK A R) was used to express the rat receptor in acini from CCK A receptor-deficient mice (CCK A R ؊/؊). Infection of CCK A R ؊/؊ acini in vitro with pAdCCK A R led to a time-dependent increase in 125 I-CCK 8 binding. The affinity for JMV-180 of the adenovirally transferred rat and the endogenous mouse CCK A receptors was not different. In native mouse acini, JMV-180 acted as a full agonist (both stimulation and inhibition of amylase release). In contrast, in mouse acini expressing pAdCCK A R JMV-180 acted as a partial agonist (only stimulation of amylase release). In addition, the pattern of protein synthesis induced by JMV-180 in CCK A R ؊/؊ mouse acini infected with AdCCK A R resembled the pattern observed in wild-type rats (lack of inhibition) rather than the respective pattern in wild-type mice (inhibition). These data suggest that species differences in the CCK A receptor of rats and mice account for the observed divergence in the acinar cell response to JMV-180.Gene transfer studies provide a powerful approach to investigate the correlation between receptor structure and function. Manipulation of the cDNA encoding a receptor followed by expression of the mutant protein in a cell model allows detailed analysis of the role of specific receptor domains and individual residues. This approach has been widely utilized and has resulted in important advancement in our understanding of G protein-coupled receptor signaling (1). However, it has become increasingly apparent that the choice of cell model used for gene transfer experiments significantly impacts the receptor properties that are observed (2). Differentiated cells express specific subsets of proteins that interact with G protein-linked receptors; examples include G proteins, arrestins, G protein receptor kinases, and RGS proteins (3). The cell model may therefore exert a major influence on the outcome of receptor structure-function studies.Recently it was suggested that interspecies variation in cellular milieu might explain observed differences between the drug-induced responses of the rat versus the mouse CCK A receptor (4). In both species, CCK A receptors bind the endogenous hormone cholecystokinin (CCK) 1 with high affinity. A similar pattern of cell signals and biological responses is in turn triggered in both rats and mice. In contrast, the CCK analog JMV-180 acts as a partial agonist in the rat and a full agonist in the mouse (5-7). Differences between partial and full CCK agonists are striking in the pancreatic acinar cell. Full agonists (e.g. CCK-8) show marked differences in biological effects at both physiologic and supra-physiologic concentrations. At physiologic concentrations, full agonists cause increases in...