Biosynthesis Of Oligomers Of Gpcrs

2.1. Intracellular Formation of GPCR Oligomers

The study of specific GPCR mutants, both naturally occurring and genetically modified, has provided a useful tool in locating the intracellular site of GPCR oligomer formation (24-29). An increasing number of reports demonstrate that co-expression of various intracellularly sequestered GPCR mutants with the corresponding wild-type receptors results in intracellular retention of the wild-type receptor. These dominant negative effects are a consequence of receptor oligomerization and provide evidence for constitu-tively formed GPCR oligomers. A physiologically relevant example of dominant negative inhibition of GPCR function is provided by the naturally occurring ccr5A32 deletion mutant of the CCR5 chemokine receptor, a coreceptor for human immunodeficiency virus (HIV) infection. This truncated nonfunctional variant of the CCR5 receptor is localized in the endo-plasmic reticulum and reduces cell surface expression of the wild-type CCR5 by oligomerization, rendering it aberrantly trapped and unable to support HIV1 infection (26). Other naturally occurring examples of dominant inhibition can be drawn from splice variants of certain GPCRs such as the gona-dotropin-releasing hormone receptor (27) and the photoreceptor rhodopsin (29). Each of these truncated receptors sequester their respective wild-type receptor in an intracellular compartment, likely by oligomerization in the ER. There are also examples of genetically derived receptor mutants that yield dominant inhibition of native receptors as a consequence of receptor-receptor interactions. Truncation mutants of the V2 vasopressin receptor have been shown to negatively regulate wild-type receptor function by forming a hetero-oligomer that is intracellularly retained (25). Similarly, point mutants of the human platelet-activating factor receptor (30) and the D2 dopamine receptor (31) have been shown to decrease binding and cell-sur face expression of the cognate wild-type receptor. Although the precise site of intracellular retention has not been conclusively determined for most of these sequestered oligomers, it indicates that oligomerization occurs prior to cell-surface expression, lending support for the constitutive oligomeric assembly of these receptors.

Several methods have been used to determine where receptor oligomers are formed in the biosynthetic pathway. Sucrose density gradient fractionation has provided a reliable means of isolating various subcellular compartments. Immunoblot analysis of these fractions has provided information regarding where GPCR oligomers are formed and how they are processed as they make their way to the plasma membrane. The advent of biophysical techniques in the study of GPCR oligomerization has provided a unique strategy for assessing the proximity of two receptors in the cell. Bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) have enabled the measurement of receptor-receptor proximity within a range of 50 to 100 angstroms, a distance that would permit receptor oligomerization. The combination of BRET or FRET and subcellular fractionation has provided a powerful tool for determining the presence of GPCR oligomers in specific organelles. BRET signals have been reported to be the highest in ER and plasma membrane-rich fractions of cells expressing oxytocin, vasopressin, and CCR5 chemokine receptor oligomers (32,33). This indicates that the earliest site of oligomer formation is the ER and that oligomeric stability is maintained during transit through the secretory pathway to the cell surface. Similar expression studies of the human complement C5a anaphylatoxin receptor have used FRET to demonstrate that these receptors also exist as oligomers in the ER, Golgi, and cell surface

(34). C5a receptor FRET signals are not affected by ligand induction, implying that GPCR oligomerization is insensitive to ligand treatment and favoring the view that oligomers are assembled in the ER. These reports corroborate well with the studies involving dominant negative receptor mutants that imply that GPCR oligomers are constitutively formed in the ER.

2.2. The Role of Glycosylation in Oligomer Formation

Most GPCRs have been shown to possess N-linked glycosylation sites in extracellular regions that serve as sites for cotranslational addition of high-mannose oligosaccharides. Mutation of these sites can result in reduced cell-surface expression of certain GPCRs, including the D5 dopamine receptor

(35) and the ATI angiotensin receptor (36), implicating a role for glycosylation in intracellular trafficking. It has been suggested that various elements, such as Rab GTPases, vesicular composition, and posttranslational modifications (like glycosylation), differentially modulate exocytosis-medi-ated transport (37). The role of glycosylation (if a role exists) in GPCR oli-gomer formation has not been clearly established. There is evidence to suggest that N-linked glycosylation-deficient mutants of the V2 vasopressin receptor (25) and the D1 dopamine receptor (Fig. 2) can form oligomeric complexes on sodium dodecyl sulfate-polyacylamide gel electrophoresis. Similar results were found in cells expressing metabotropic glutamate receptor 1a that were pre-incubated with glycosylation inhibitors such as tunicamycin (38). Although these examples suggest that glycosylation has no role in GPCR oligomerization, studies of adrenergic receptor (AR) oligomers challenge this notion and implicate receptor-specific modulation by glycosylation. The decreased ability to co-immunoprecipitate differentially tagged glycosylation mutants of the Pi-AR compared to the wild-type receptor provides evidence that in this case, glycosylation may actually be required for receptor homo-oligomerization (39). Conversely, it was demonstrated that the same glycosylation mutant of the P1-AR could heterodimerize more efficiently with wild-type a2A- AR than with wild-type P1-AR (40). The reciprocal experiment with a glycosylation-impaired a2A-AR yielded similar results, suggesting that glycosylation may sterically hinder the efficiency of these ARs to hetero-oligomerize. Thus, it appears that abolishing glycosylation in the P1-AR has differential effects on its propensity to homo-and hetero-oligomerize.

2.3. Resident ER Chaperones Aid in Receptor Oligomerization

The processing of proteins in the ER involves rigorous quality control mechanisms to ensure that the proteins adopt a conformation compatible for proper trafficking through the distal secretory pathway (41). Because of the hydrophobic nature of many nascent proteins, the cell employs ER-resident chaperone proteins that function within the framework of a quality control mechanism to monitor the folding of functional oligomeric proteins, thus ensuring that they do not aggregate or misfold.

Constitutive oligomeric assembly of glycoproteins in the cell, including receptors and ion channels, is tightly regulated by ER-resident proteins known as molecular chaperones (42-45). These proteins function by binding to and assisting the folding kinetics of polypeptides as they are extruded from the ER (Fig. 1). Molecular chaperones can be classified into four main families: the heat shock proteins (including Hsp40, Hsp70, and Hsp90), the lectin family of chaperones (including calnexin and calreticulin), the peptidyl-prolyl isomerases, and the thiol-disulphide-oxidoreductases (46). An elegant

Fig. 2. The wild-type D1 dopamine receptor (WT-D1) exists as dimeric and higher order oligomeric forms. The glycosylation-deficient mutant (D1-glyc def.) has alanine mutations at N5 and N175 and exhibits a similar expression pattern, with a reduction in size of all species corresponding to the expected size of the unglycosylated D1 receptor. Monomeric species are dissociation products resulting from treatment with reducing agents.

Fig. 2. The wild-type D1 dopamine receptor (WT-D1) exists as dimeric and higher order oligomeric forms. The glycosylation-deficient mutant (D1-glyc def.) has alanine mutations at N5 and N175 and exhibits a similar expression pattern, with a reduction in size of all species corresponding to the expected size of the unglycosylated D1 receptor. Monomeric species are dissociation products resulting from treatment with reducing agents.

study exemplifying the role of chaperones in oligomeric receptor assembly involves the single-transmembrane-spanning human insulin receptor (HIR), which is expressed at the cell surface as a functional ER-derived homodimer (42). HIR maturation involves the cotranslational trimming of three glucose residues by glucosidase I and II to a single, terminal glucose on high-man-nose-type oligosaccharides. The resulting monoglucosylated core glycan serves as a substrate for binding to calnexin and calreticulin, which is required for proper folding and dimerization of nascent receptor monomers. The addition of glucose trimming inhibitors such as castanospermine prevents the binding of these chaperones to the HIR, resulting in premature processing manifested as accelerated dimerization and misfolded oligomeric assembly (42). Therefore, the HIR requires chaperone association to maintain oligomer fidelity, possibly by sterically masking hydrophobic interfaces that otherwise would cause aggregation of the nascent monomeric protein.

Similar detailed studies with GPCRs have not been reported, but there is evidence suggesting that molecular chaperones may participate in GPCR oli-gomeric assembly in the ER. Both the V2 vasopressin receptor and the gona-dotropin-releasing hormone receptor have been shown to form oligomers constitutively (27,32,47) and to interact with calnexin (48,49). The thyrotropin receptor (TSHR) has been reported to interact with BiP (a prototypic Hsp70), calnexin, and calreticulin in the ER (50), and each interaction has unique effects on receptor synthesis and folding. Calnexin and calreticulin appear to stabilize the TSHR and blunt degradation of newly synthesized receptors, whereas association with BiP destabilizes the receptor and promotes proteasomal degradation. As a result, the maturation of TSHR and its ultimate cellular fate is highly dependent on which chaperone system participates in folding after protein synthesis. The TSHR has also been shown to form constitutive oligomers, as detected by FRET and by immunodetection of oligomers in detergent-solubilized thyroid membranes (51,52). Therefore, because of the role of lectin chaperones in insulin receptor oligomer maturation, it is conceivable that calnexin and calreticulin promote maturation of TSHR by helping to mediate proper oligomeric assembly in the ER. Evidence for the self-dimer-ization of calreticulin (53), calnexin (54), and specific HSP90 chaperones (55,56) suggests a mechanism by which chaperone dimers bind to and facilitate the folding of GPCR oligomers. Thus, the ubiquitous role of chaperones in general oligomeric assembly of proteins implicates a functional role for molecular chaperones in GPCR oligomer formation.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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