Glycosylation of high-affinity thrombin receptors appears necessary for thrombin binding

Frost, Gloria H.; Bergmann, John S.; Carney, Darrell H.

doi:10.1016/s0006-291x(05)81299-9

Cited by 10 publications

(3 citation statements)

References 21 publications

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“…Perhaps correctly targeted mRNA translation allows for appropriate post-translational modifications to occur. For example, most G-protein-coupled receptors are glycosylated at N-terminal asparagine residues and this modification can contibute significantly to the molecular mass of the mature receptor protein [46,47]. The Ang II receptor is glycosylated [16] and, although the function of this modification is not known, it may allow for correct folding and\or insertion of the receptor into the membrane.…”

Section: Discussionmentioning

confidence: 99%

Functional role for the angiotensin II receptor (AT1A) 3′-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins

Thekkumkara

Gruber

Motel

et al. 1998

Biochemical Journal

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We demonstrate a functional role for the 3ʹ-untranslated region (3ʹ-UTR) of the angiotensin II (Ang II) receptor subtype AT1A mRNA in Chinese hamster ovary (CHO-K1) cells by stably transfecting the coding region of the receptor gene with or without the 845 bp 3ʹ-UTR. Two cell lines expressing similar levels of cell-surface receptors (with 3ʹ-UTR, Bmax = 571 fmol/mg protein; without 3ʹ-UTR, Bmax = 663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3ʹ-UTR, Kd = 0.83 nM; without 3ʹ-UTR, Kd = 0.82 nM), and binding studies with 125I-labelled Ang II in the presence of GTP[S] demonstrated that both coupled to heterotrimeric G-proteins. Despite these similarities, significant differences were observed for receptor-mediated cell signalling pathways. In cells without the 3ʹ-UTR, Ang II stimulated an increase in cAMP accumulation (11-fold above control) and in cells with the 3ʹ-UTR no stimulation was observed, which was consistent with previous observations in most endogenous Ang II receptor (AT1)-expressing cells. Activation of cAMP by Ang II in cells without the 3ʹ-UTR correlated with an inhibition of DNA synthesis, determined by [3H]thymidine incorporation. Ang II-mediated responses were blocked by EXP3174, a selective non-peptide receptor antagonist. We also observed differences in the transient profiles of intracellular calcium between cells with and without the 3ʹ-UTR in response to Ang II. In cells with the 3ʹ-UTR, a sustained level of intracellular calcium was observed after Ang II stimulation, whereas cells without the 3ʹ-UTR displayed a full return to basal level within 50 s of Ang II treatment. Even though the expressed exogenous gene is under the control of a constitutively expressing promoter (cytomegalovirus promoter), Northern-blot analysis revealed a considerably greater accumulation of AT1A mRNA in cells without the 3ʹ-UTR compared with cells with the 3ʹ-UTR. Analysis of the decay rate of the AT1A mRNA in cells with and without the 3ʹ-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3ʹ-UTR, identifying a role for the 3ʹ-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3ʹ-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3ʹ-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3ʹ-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3ʹ-UTR-binding assays, electrophoretic mobility-shift assays and UV crosslinking studies were performed. A major cellular protein of 55 kDa was identified, which specifically interacted with the 3ʹ-UTR. Our data suggest that the 3ʹ-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3ʹ-UTR by RNA-binding proteins.

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Section: Discussionmentioning

confidence: 99%

Functional role for the angiotensin II receptor (AT1A) 3′-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins

Thekkumkara

Gruber

Motel

et al. 1998

Biochemical Journal

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“…This is intriguing considering that more than 50% of the molecular mass of the extracellular domain of TrkB in vivo is made up of carbohydrate moieties. It has been previously shown for the interaction of several ligands with their receptors that glycosylation is not necessarily an important determinant of these processes (Schwarz et al, 1991;Sairam, 1989), whereas in other cases (Frost et al, 1991;Moller et al, 1993) it seems to contribute considerably to ligand-binding affinity and/or specificity.…”

Section: Resultsmentioning

confidence: 99%

Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 bind to a single leucine-rich motif of TrkB

Windisch¹,

Marksteiner²,

Lang³

et al. 1995

Biochemistry

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TrkB is a member of the Trk family of neurotrophin receptors. Its extracellular domain exhibits the same modular structure found in its homologs, TrkA and TrkC, consisting of an N-terminal LRM3 cassette and two immunoglobulin-like modules (Ig2 domain) adjacent to the membrane. The LRM3 cassette comprises two cysteine-rich clusters framing a tandem array of three leucine-rich motifs (LRMs). On the basis of the recent identification of a nerve growth factor (NGF) binding site within TrkA, the ability of the different structural entities within the extracellular domain of TrkB to bind the various neurotrophins was determined by using a recombinant receptor approach. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) bound to the LRM3 cassette of TrkB, whereas NGF did not. These binding characteristics evidently reflect in vivo specificities. A more precise mapping of the region(s) responsible for binding BDNF, NT-3, and NT-4 identified the second leucine-rich motif of TrkB as a functional unit capable of binding all three neurotrophins. The affinities and kinetics that this short stretch of amino acids exhibited with respect to the different neurotrophins were clearly akin to those observed for cells ectopically expressing TrkB receptors. With 24 amino acids determining the affinities and kinetics of the interactions with three different partners, the leucine-rich motif is strongly established as one of the most potent and flexible protein--protein interaction motifs.

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“…Many GPCRs contain at least one consensus site for N -linked glycosylation that may confer some functional properties. For example, glycosylation of thrombin receptors appears to be necessary for thrombin binding whereas glycosylation of the β2-adrenergic receptor has no effect on ligand binding but is necessary for normal G protein coupling [22, 23]. The availability of an excellent radioligand binding assay for P2Y 1 receptors permits quantification of functional P2Y 1 receptor binding sites at each step of purification using the radiolabeled antagonist, [ 3 H]MRS2279 [24].…”

Section: Receptor Purificationmentioning

confidence: 99%

Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins

Bodor

Waldo

Blaesius

et al. 2004

Purinergic Signalling

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P2Y receptors are G protein coupled receptors that respond to extracellular nucleotides to promote a multitude of signaling events. Our laboratory has purified several P2Y receptors with the goal of providing molecular insight into their: (1) ligand binding properties, (2) G protein signaling selectivities, and (3) regulation by RGS proteins and other signaling cohorts. The human P2Y1 receptor and the human P2Y12 receptor, both of which are intimately involved in ADP-mediated platelet aggregation, were purified to near homogeneity and studied in detail. After high-level expression from recombinant baculovirus infection of Sf9 insect cells, approximately 50% of the receptors were successfully extracted with digitonin. Purification of nearly homogeneous epitope-tagged P2Y receptor was achieved using metal-affinity chromatography followed by other traditional chromatographic steps. Yields of purified P2Y receptors range from 10 to 100 μg/l of infected cells. Once purified, the receptors were reconstituted in model lipid vesicles along with their cognate G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase assays demonstrated the functional activity of the reconstituted purified receptor. We have utilized this reconstitution system to assess the action of various nucleotide agonists and antagonists, the relative G protein selectivity, and the influence of other proteins, such as phospholipase C, on P2Y receptor-promoted signaling. Furthermore, we have identified the RGS expression profile of platelets and have begun to assess the action of these RGS proteins in a reconstituted P2Y receptor/G protein platelet model.

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Glycosylation of high-affinity thrombin receptors appears necessary for thrombin binding

Cited by 10 publications

References 21 publications

Functional role for the angiotensin II receptor (AT1A) 3′-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins

Functional role for the angiotensin II receptor (AT1A) 3′-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins

Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 bind to a single leucine-rich motif of TrkB

Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins

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