Biochemical and cell biological mechanisms of cholecystokinin receptor regulation.

Regulation of the cholecystokinin receptor is accomplished by biochemical and cell biological mechanisms. The major mechanism for biochemical regulation involves phosphorylation of serine and threonine residues within the receptor's intracellular third loop and carboxyl-terminal tail. This form of rapid desensitization is achieved by protein kinase C, a kinase activated in the normal signaling cascade of this Gq-coupled receptor, and/or a member of the G protein-coupled receptor kinase family that recognizes the active conformation of the receptor.

Mapping the architecture of secretin receptors with intramolecular fluorescence resonance energy transfer using acousto-optic tunable filter-based spectral imaging.

The molecular structure and agonist-induced conformational changes of class II G protein-coupled receptors are poorly understood. In this work, we developed and characterized a series of dual cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged secretin receptor constructs for use in various functional and fluorescence analyses of receptor structural variants. CFP insertions within the first or second intracellular loop domains of this receptor were tolerated poorly or partially, respectively, in receptors tagged with a carboxyl-terminal yellow fluorescent protein that itself had no effect on secretin binding or cAMP production.

Secretin receptor oligomers form intracellularly during maturation through receptor core domains.

Oligomerization of numerous G protein-coupled receptors has been documented, including the prototypic family B secretin receptor. The clinical significance of oligomerization of this receptor became clear with the recent observation that a misspliced form present in pancreatic cancer could associate with the wild-type receptor and act as a dominant negative inhibitor of its normal growth inhibitory function. Our goal was to explore the molecular mechanism of this interaction using bioluminescence (BRET) and fluorescence (FRET) resonance energy transfer and fluorescence microscopy with a variety of receptor constructs tagged with luciferase or cyan or yellow fluorescent proteins.

Constitutive formation of oligomeric complexes between family B G protein-coupled vasoactive intestinal polypeptide and secretin receptors.

Formation of oligomeric complexes of family A G protein-coupled receptors has been shown to influence their function and regulation. However, little is known about the existence of such complexes for family B receptors in this superfamily. We previously used bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic family B secretin receptor forms ligand-independent oligomeric complexes.

Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase.

The H2O2 byproduct of fatty acid catabolism in plant peroxisomes is removed in part by a membrane-associated antioxidant system that involves both an ascorbate peroxidase and a monodehydroascorbate reductase (MDAR). Despite descriptions of 32-kDa MDAR polypeptides in pea and castor peroxisomal membranes and cDNA sequences for several 'cytosolic' MDARs, the genetic and protein factors responsible for peroxisomal MDAR function have yet to be elucidated. Of the six MDAR polypeptides in the Arabidopsis proteome, named AtMDAR1 to AtMDAR6 in this study, 47-kDa AtMDAR1 and 54-kDa AtMDAR4 possess amino acid sequences that resemble matrix (PTS1) and membrane peroxisomal targeting signals, respectively.

Paired cysteine mutagenesis to establish the pattern of disulfide bonds in the functional intact secretin receptor.

The amino-terminal domain of class B G protein-coupled receptors contains six conserved cysteine residues involved in structurally and functionally critical disulfide bonds. The mapping of these bonds has been unclear, with one pattern based on biochemical and NMR structural characterizations of refolded, nonglycosylated amino-terminal fragments, and another pattern derived from functional characterizations of intact receptors having paired cysteine mutations. In the present study, we determined the disulfide bonding pattern of the prototypic class B secretin receptor by applying the same paired cysteine mutagenesis approach and confirming the predicted bonding pattern with proteolytic cleavage of intact functional receptor.

Peroxisomal ascorbate peroxidase resides within a subdomain of rough endoplasmic reticulum in wild-type Arabidopsis cells.

Previously we reported (R.T. Mullen, C.

Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal ER.

Peroxisomal ascorbate peroxidase (APX) sorts indirectly via a subdomain of the ER (peroxisomal ER) to the boundary membrane of peroxisomes in tobacco Bright Yellow 2 cells. This novel subdomain characteristically appears as fluorescent reticular/circular compartments distributed variously in the cytoplasm. Further characterizations are presented herein.

Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain.

Peroxisomal ascorbate peroxidase (APX) (EC 1.11.1.

Peroxisomal membrane ascorbate peroxidase is sorted to a membranous network that resembles a subdomain of the endoplasmic reticulum.

The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD(+) and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3'-dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes.