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At least 343 records · Page 19Linked to original sources
PubMed2003

Serotonin 5-HT(2B) receptor loss of function mutation in a patient with fenfluramine-associated primary pulmonary hypertension.

OBJECTIVE: Appetite-suppressant drug fenfluramine is implicated in primary pulmonary hypertension (PPH) but the molecular pathways that mediate this effect are unknown. A mouse model incriminates the serotonin 5-HT(2B) receptor but contrasts with other models where this receptor has been shown to mediate pulmonary arterial relaxation via nitric oxide production. METHODS: We analyzed the human 5-HT(2B) gene in 10 patients with appetite-suppressant drug-associated PPH. RESULTS: A mutation causing premature truncation of the protein product was found in one patient. The mutation was not found in 80 control subjects and no 5-HT(2B) mutation was found in 18 PPH patients not associated with appetite-suppressants. Functional analysis of the transfected receptor expressed either transiently in COS cells or stably in CHO cells demonstrated that the mutated receptor fails to activate the second messenger inositol-phosphates cascade and subsequent intracellular calcium release, in spite of normal expression at the cell membrane. The mutated receptor had no constitutive activity, and produced no dominant negative effect on the wild-type receptor. CONCLUSION: Loss of serotonin 5-HT(2B) receptor function may predispose to fenfluramine-associated PPH in man.

Animals↗
PubMed2005

Assays for interaction between Rab7 and oxysterol binding protein related protein 1L (ORP1L).

ORP1L belongs to the recently described family of human oxysterol binding protein homologues. We have previously shown that ORP1L localizes to late endosomes. In this chapter we describe methods that have been used to investigate the functional link of ORP1L with the protein machinery regulating late endosomal membrane trafficking. Co-immunoprecipitation, COS cell two-hybrid, and pull-down assays were applied to demonstrate a physical interaction between ORP1L and the late endosomal small GTPase Rab7. With these methods we were able to map the Rab7-binding determinant of ORP1L to the amino-terminal ankyrin repeat region (aa 1-237) and show that the interaction is preferentially with the GTP-bound form of Rab7. Furthermore, we describe approaches based on transient transfection and confocal immunofluorescence microscopy, which were employed to study the effect of this amino-terminal ORP1L fragment on late endosome morphology. The ankyrin repeat fragment induces juxtanuclear clustering of late endosomes, dependent on an intact microtubule network. When it is coexpressed with the dominant inhibitory Rab7 mutant T22N, the clustering is inhibited, suggesting that the effect involves interaction of the fragment with active Rab7.

Animals↗
PubMed2003

CLIC6, a member of the intracellular chloride channel family, interacts with dopamine D(2)-like receptors.

To identify proteins interacting with the C-terminal cytoplasmic tail of the dopamine D(3) receptor (D(3)R), we used the two-hybrid system to screen a rat brain cDNA library. We isolated three partial cDNAs encoding, respectively, for the MUPP1 multi-PDZ protein, for the N-terminal region of radixin, for GIPC and for a 160-amino acid open reading frame sharing high homology with the human CLIC6, also identified as parchorin in rabbit. In the two-hybrid system, CLIC6 was also able to interact with the D(2)R and D(4)R. The interaction between D(3)R and CLIC6 was confirmed by the use of a GST-D(3)R C-terminus fusion protein and COS cell extracts transiently expressing epitope-tagged CLIC6. In adult rat brain, CLIC6 mRNA expression was restricted to the choroid plexus, the striatal proliferative subventricular zone and the cerebellum where it is co-expressed with the D(3)R in the Purkinje cells of the lobules IX and X. CLIC6 mRNA was also detected in the pituitary in the posterior lobe and in cells co-expressing the D(2)R at the border between the intermediate and anterior lobes. In transfected HEK293 cells, D(3)R and CLIC6 co-localized at the plasma-membrane. No effect of CLIC6 transfection was observed on either intracellular chloride concentration or D(3)R/D(2)R-mediated response. In two-hybrid system, CLIC6 also interacted with MUPP1 and radixin but not GIPC, suggesting it could take part in a complex with D(2)-like receptors, not only by direct interaction with their C-termini, but also through interactions with scaffolding proteins.

Amino Acid Sequence↗
PubMed1999

Phosphorylation of the DNA repair protein APE/REF-1 by CKII affects redox regulation of AP-1.

The DNA repair protein apurinic endonuclease (APE/Ref-1) exerts several physiological functions such as cleavage of apurinic/apyrimidinic sites and redox regulation of the transcription factor AP-1, whose activation is part of the cellular response to DNA damaging treatments. Here we demonstrate that APE/Ref-1 is phosphorylated by casein kinase II (CKII). This was shown for both the recombinant APE/Ref-1 protein (Km=0.55 mM) and for APE/Ref-1 expressed in COS cells. Phosphorylation of APE/Ref-1 did not alter the repair activity of the enzyme, whereas it stimulated its redox capability towards AP-1, thus promoting DNA binding activity of AP-1. Inhibition of CKII mediated phosphorylation of APE/Ref-1 blocked mutagen-stimulated increase in AP-1 binding. It also abrogated the induction of c-Jun protein and rendered cells more sensitive to induced DNA damage. Thus, phosphorylation of APE/Ref-1 appears to be involved in regulating the different physiological activities of the enzyme. CKII mediated phosphorylation of APE/Ref-1 and concomitant increase in AP-1 binding activity appears to be a novel mechanism of cellular stress response, forcing transcription of AP-1 target gene(s) the product(s) of which may exert protective function.

Animals↗
PubMed2002

N-glycosylation is required for full enzymic activity of the murine galactosylceramide sulphotransferase.

3- O -Sulphogalactosylceramide (sulphatide) is a major lipid component of myelin membranes, and is required for proper myelin formation. Sulphatide is synthesized in the Golgi apparatus by galactosylceramide sulphotransferase (CST; EC 2.8.2.11). Murine and human CSTs contain two putative N-glycosylation sites (Asn-66 and Asn-312). The second site is conserved among all galactose 3-O-sulphotransferases cloned to date. In order to study the functional relevance of N-glycosylation, we generated epitope-tagged CST and soluble Protein A-CST fusion proteins lacking both N-glycosylation sites, separately or in combination. Our results show that both sites are glycosylated when CST is expressed in Chinese hamster ovary (CHO) or COS cells. Moreover, transfecting CST mutants lacking both N-glycosylation sites, or only Asn-312, reduced significantly the amount of sulphatide synthesized, whereas substituting Asn-66 with a glutamine residue did not. In contrast, activity in vitro was reduced by approx. 50% in the Asn-66-->Gln (N66Q) mutant, and was almost undetectable in N312Q and N66/312Q transfectants. Furthermore, soluble Protein A-CST expressed in the presence of tunicamycin was almost inactive, and accumulated in transfected cells. Expression of fully active CST in a CHO-glycosylation mutant lacking N-acetylglucosaminyltransferase I demonstrated that condensation of the N-linked pentamannosyl-core structure is sufficient to form a fully active enzyme.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of a vesicular monoamine transporter by casein kinase II.

The vesicular monoamine transporters (VMATs) package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. One isoform occurs in the adrenal gland (VMAT1) and another in the brain (VMAT2). To assess their potential for regulation, we have investigated the phosphorylation of the VMATs. Using heterologous expression in Chinese hamster ovary, PC12, and COS cells, we find that rat VMAT2, but not VMAT1, is constitutively phosphorylated. Phosphoamino acid analysis indicates that this phosphorylation occurs on serine residues, and the analysis of VMAT1-VMAT2 chimeras and site-directed mutagenesis localize the phosphorylation sites to serines 512 and 514 at the carboxyl terminus of VMAT2. Since these residues occur in an acidic region, we tested the ability of the acidotropic kinases casein kinase I (CKI) and casein kinase II (CKII) to phosphorylate bacterial fusion proteins containing the carboxyl terminus of VMAT2. Purified CKI and CKII phosphorylate the wild-type carboxyl terminus of VMAT2, but not a double mutant with both serines 512 and 514 replaced by alanine. The protein kinase inhibitor CKI-7 and unlabeled GTP both block in vitro phosphorylation by cell homogenates, indicating a role for CKII and possibly CKI in vivo. Both kinases phosphorylate the VMAT2 fusion protein to a much greater extent than a similar fusion protein containing the carboxyl terminus of VMAT1, consistent with differential phosphorylation of the two transporters observed in intact cells. These results provide the first demonstration of phosphorylation of a vesicular neurotransmitter transporter and a potential mechanism for differential regulation of the two VMATs.

Adenosine Triphosphate↗
PubMed1999

Chicken macrophage stimulating protein is a ligand of the receptor protein-tyrosine kinase Sea.

Affinity chromatography, employing the extracellular domain of the Sea receptor, was used to enrich Sea-binding proteins from chicken serum. One isolated protein bound both a Sea-immunoglobulin fusion protein and an antisera raised against murine macrophage stimulating protein. Amino-terminal sequencing of the dual-reactive protein yielded sequences which were identical to the predicted alpha and beta subunits of chicken macrophage stimulating protein. The partially purified chicken macrophage stimulating protein caused autophosphorylation of the Sea receptor. Previous work showed that recombinant expression of fully activatible human or mouse macrophage stimulating protein required a specific Cys to Ala substitution (Wahl, R. C., Costigan, V. J., Batac, J. P., Chen, K., Cam, L., Courchesne, P. L., Patterson, S. D. Zhang, K., and Pacifici, R. E. (1997) J. Biol. Chem. 272, 1-4). Therefore, we expressed both the wild type and the specific Cys to Ala form of chicken macrophage stimulating protein as recombinant proteins. After proteolytic activation, only conditioned media from COS cells transfected with the C665A chicken macrophage stimulating protein, but not from wild type chicken macrophage-stimulating protein, or control vector, was detected by the Sea-immunoglobulin fusion protein in Western blotting experiments. Conditioned media containing the C665A chicken macrophage-stimulating protein readily caused Sea phosphorylation, while conditioned media containing the wild type chicken macrophage-stimulating protein was only effective at inducing receptor phosphorylation at high concentrations. In addition to receptor phosphorylation, the C665A chicken macrophage-stimulating protein induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.

Animals↗
PubMed1999

The release of acetylcholine receptor inducing activity (ARIA) from its transmembrane precursor in transfected fibroblasts.

Acetylcholine receptor inducing activity (ARIA) is made by motoneurons and is released at the neuromuscular synapse to stimulate the synthesis of acetylcholine receptors by skeletal muscle. ARIA is derived from a transmembrane precursor (pro-ARIA) via proteolytic cleavage of the ectodomain. We studied requirements in the amino acid sequence at the cleavage site with various substitution and deletion mutations. Wild type (WT) and mutant proteins were transiently expressed in COS cells, and release of ARIA into the conditioned medium was measured by tyrosine phosphorylation of its receptor, p185, in L6 cells. Removal of all potential cleavage sites between the extracellular epidermal growth factor domain and the transmembrane domain by substitution and small deletions (<11 amino acid residues out of 21) did not significantly reduce ARIA release, whereas larger deletions abolished it. We propose that cleavage occurs independently of amino acid sequence at a short distance from the epidermal growth factor domain, unless sterically hindered by the nearby secondary structure. A mutant with shorter cytoplasmic domain ("c" isoform) released significantly less ARIA than the WT ("a" isoform), suggesting that the c isoform may be suitable for signaling through direct cell-cell contact. Alternatively, proteolytic conversion of the a isoform to the c isoform may rapidly down-regulate release of ARIA.

Amino Acid Sequence↗
PubMed1999

The processing of ligands by the class A scavenger receptor is dependent on signal information located in the cytoplasmic domain.

The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser(21) increased acetyl-LDL metabolism, whereas inactivation of the site at Ser(49) reduced acetyl-LDL uptake. The increase in uptake by the Ser(21) mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser(49) did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, beta-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp(25) inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand.

Animals↗
PubMed2000

Polymorphic deletion of three intracellular acidic residues of the alpha 2B-adrenergic receptor decreases G protein-coupled receptor kinase-mediated phosphorylation and desensitization.

A polymorphic variant of the human alpha(2B)-adrenergic receptor (alpha(2B)AR), which consists of a deletion of three glutamic acids (residues 301-303) in the third intracellular loop was found to be common in Caucasians (31%) and to a lesser extent in African-Americans (12%). The consequences of this deletion were assessed by expressing wild-type and the Del301-303 receptors in Chinese hamster ovary and COS cells. Ligand binding was not affected, although a small decrease in coupling efficiency to the inhibition of adenylyl cyclase was observed with the mutant. The deletion occurs within a stretch of acidic residues that is thought to establish the milieu for agonist-promoted phosphorylation and desensitization of the receptor by G protein-coupled receptor kinases (GRKs). Agonist-promoted phosphorylation studies carried out in cells coexpressing the alpha(2B)ARs and GRK2 revealed that the Del301-303 receptor displayed approximately 56% of wild-type phosphorylation. Furthermore, the depressed phosphorylation imposed by the deletion was found to result in a complete loss of short term agonist-promoted receptor desensitization. Thus the major phenotype of the Del301-303 alpha(2B)AR is one of impaired phosphorylation and desensitization by GRKs, and thus the polymorphisms renders the receptor incapable of modulation by this key mechanism of dynamic regulation.

Adenylyl Cyclases↗
PubMed2001

The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling.

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX(3)C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.

Animals↗
PubMed2006

The retention factor p11 confers an endoplasmic reticulum-localization signal to the potassium channel TASK-1.

The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.

14-3-3 Proteins↗
PubMed2000

Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of G(i/o)-coupled receptors: changes in detergent solubility are in correlation with onset of adenylyl cyclase superactivation.

Prolonged G(i/o) protein-coupled receptor activation has been shown to lead to receptor internalization and receptor desensitization. In addition, it is well established that although acute activation of these receptors leads to inhibition of adenylyl cyclase (AC), long-term activation results in increased AC activity (especially evident on removal of the inhibitory agonist), a phenomenon defined as AC superactivation or sensitization. Herein, we show that chronic exposure to agonists of G(i)-coupled receptors also leads to a decrease in cholate detergent solubility of G protein subunits, and that antagonist treatment after such chronic agonist exposure leads to a time-dependent reversal of the cholate insolubility. With Chinese hamster ovary and COS cells transfected with several G(i/o)-coupled receptors (i.e., mu- and kappa-opioid, and m(4)-muscarinic), we observed that although no overall change occurred in total content of G(alphai)- and beta(1)-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% cholate to solubilize G(betagamma) as well as G(alphai). This solubility shift is exclusively observed with G(alphai), and was not seen with G(alphas). The disappearance and reappearance of G(alphai) and G(betagamma) subunits from and to the detergent-soluble fractions occur with similar time courses as observed for the onset and disappearance of AC superactivation. Lastly, pertussis toxin, which blocks acute and chronic agonist-induced AC inhibition and superactivation, also blocks the shift in detergent solubility. These results suggest a correlation between the solubility shift of the heterotrimeric G(i) protein and the generation of AC superactivation.

Adenylate Cyclase Toxin↗
PubMed1997

Growth hormone-induced tyrosyl phosphorylation and deoxyribonucleic acid binding activity of Stat5A and Stat5B.

GH is known to activate JAK2 tyrosine kinase and members of the Stat family of transcription factors, including Stats 1, 3, and 5. The recent observation that at least two Stat5 proteins (Stat5A and Stat5B) exist in mouse and human, raises the question of whether GH activates both Stat5A and Stat5B and, if so, whether the requirements for activation are the same. An initial report investigating this issue demonstrated GH-dependent activation of Stat5A but not Stat5B. In this paper, we demonstrate (in COS cells expressing rat GH receptor (rGHR) and either Stat5A or Stat5B, 3T3-F442A fibroblasts, and CHO cells expressing rGHR) that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B. Similar time courses of phosphorylation were observed for the two proteins. Interestingly, the pattern of observed bands differs for the two forms of Stat5. Two closely migrating Stat5A bands can be detected in cells treated with or without GH. Both of these bands become tyrosyl phosphorylated in response to GH. Three species of Stat5B are observed in untreated cells. An additional, more slowly migrating Stat5B band, appears upon treatment with GH. The three more slower migrating Stat5B bands observed in response to GH contain phosphorylated tyrosyl residues. We further demonstrate that GH induces binding of Stat5A and Stat5B, as well as Stat1, to the GAS-like element in the beta-casein promoter. We and others have demonstrated previously that specific regions of GHR are required for GH-dependent activation of what is here identified as Stat5B. To gain insight into the mechanism by which GH promotes tyrosyl phosphorylation of Stat5A, GH-dependent tyrosyl phosphorylation of Stat5A was examined in CHO cells expressing truncated and mutated rGHR. The results indicate that Stat5A and Stat5B require the same regions of rGHR for maximal activation by GH: the C-terminal half of the cytoplasmic domain; tyrosines 333 and/or 338 in the N-terminal half of the cytoplasmic domain; and the regions required for JAK2 activation. To dissect further the mechanism by which GH activates Stat5A and B, the requirement for JAK2 in GH-dependent Stat5 tyrosyl phosphorylation was assessed using JAK2-deficient cells expressing GHR (gamma2A-GHR) and the wild-type parental cell line expressing GHR (2C4-GHR). GH-induced tyrosyl phosphorylation of Stat5B in 2C4-GHR cells but not in the JAK2 deficient, gamma2A-GHR cells, indicating that JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B. Western blotting revealed that Stat5A is not expressed in this cell type. Taken together, these findings suggest that: 1) GH activates both Stat5A and Stat5B in several cell types; 2) the pattern of bands observed differs for Stat5A and Stat5B; 3) GH-dependent tyrosyl phosphorylation of Stat5A requires specific regions of GHR, and these requirements are the same as for Stat5B; and 4) JAK2 kinase is required for GH-dependent tyrosyl phosphorylation of Stat5B and, most likely, Stat5A.

Animals↗
PubMed1983

Expression of chimeric genes in the early region of SV40.

Chimeric genes have been constructed by inserting foreign gene sequences in the early region of SV40. The genes contained the first exon of the SV40 large T gene with 180 bp of its intron and either the third exon of the rat preproinsulin gene II with 488 bp of its large intron or the third exon of the mouse beta globin gene with 63 bp of its intron. The chimeric genes contained a 5' splicing site (SS) from SV40 and a 3' SS from the inserted gene. Both the preproinsulin and the globin insertions contained a polyadenylation signal. The SV40 early poly(A) addition signal was also retained. High-titer virus stocks were obtained when the recombinants, which contained SV40 origin of replication and the entire late region, were used to transfect a cloned line of COS cells (COS-M6). These stocks typically contained no detectable wild-type virus. RNA mapping demonstrated the following: (a) The SV40-rat preproinsulin chimeric RNA was initiated at the SV40 early promoter, spliced from the SV40 5' SS to the rat preproinsulin 3' SS, and polyadenylated solely at the SV40 poly(A) addition signal. (b) The SV40-mouse beta globin chimeric RNA was initiated at the SV40 early promoter, spliced from the 5' SS to the mouse beta globin 3' SS, and polyadenylated at the mouse beta globin poly(A) site. The chimeric RNAs were overproduced, owing to low levels of T antigen in the COS-M6 cells, which did not completely repress transcription from the early region. Fusion proteins of 15,500 molecular weight resulted from expression in vivo of the SV40-rat preproinsulin chimeric gene and of 11,500 molecular weight for the SV40-mouse beta globin chimeric gene. The molecular weights of the proteins suggested that they were initiated at the early SV40 AUG and that translation continued across the chimeric splice sites. The chimeric proteins were also overproduced.

Animals↗
PubMed1997

Synergistic regulation of beta2-adrenergic receptor sequestration: intracellular complement of beta-adrenergic receptor kinase and beta-arrestin determine kinetics of internalization.

Two of the common mechanisms regulating G protein-coupled receptor (GPCR) signal transduction are phosphorylation and sequestration (internalization). Agonist-mediated receptor phosphorylation by the beta-adrenergic receptor kinase (betaARK) facilitates subsequent interaction with an arrestin protein, resulting in receptor desensitization. Studies of the beta2-adrenergic receptor (beta2AR) receptor in human embryonic kidney (HEK) 293 cells indicate that betaARK and arrestin proteins (beta-arrestins) also regulate sequestration. Consistent with this notion, we show in HEK 293 cells that reduction in or removal of the ability of the beta2AR to be phosphorylated by betaARK or to interact normally with beta-arrestin substantially reduces agonist-mediated sequestration. To evaluate betaARK and beta-arrestin regulation of beta2AR sequestration, we examined the relationship between betaARK and/or beta-arrestin expression and beta2AR sequestration in a variety of cultured cells, including HEK 293, COS 7, CHO, A431, and CHW. COS cells had both the lowest levels of endogenous beta-arrestin expression and beta2AR sequestration, whereas HEK 293 had the highest. Overexpression of beta-arrestin, but not betaARK, in COS cells increased the extent of wild-type beta2AR sequestration to levels observed in HEK 293 cells. However, a betaARK phosphorylation-impaired beta2AR mutant (Y326A) required the simultaneous overexpression of both betaARK and beta-arrestin for this to occur. Among all cell lines, sequestration correlated best with the product of betaARK and beta-arrestin expression. Moreover, an agonist-mediated translocation of wild-type beta2AR and endogenous beta-arrestin 2 to endocytic vesicles prepared from CHO fibroblasts was observed. These data suggest not only that the complement of cellular betaARK and arrestin proteins synergistically regulate beta2AR sequestration but also that beta-arrestins directly regulate beta2AR trafficking as well as desensitization.

Animals↗
PubMed1994

Palmitoylation of multiple Src-family kinases at a homologous N-terminal motif.

We have recently identified a novel N-terminal cysteine-containing motif which specifies the palmitoylation of several G-protein alpha-subunits [Parenti, Viganó, Newman, Milligan and Magee (1993) Biochem. J. 291, 349-353]. A related motif occurs at the N-terminus of members of the Src family of protein tyrosine kinases except for Src itself and Blk. We have investigated whether the Src, Fyn, Yes and Lck gene products are palmitoylated. Src was not labelled with [3H]palmitate when endogenously expressed in COS cells. In contrast, endogenous Yes immunoprecipitated from COS cells was palmitoylated. Fyn was palmitoylated in insect cells infected with a recombinant baculovirus and the palmitoylation was independent of protein synthesis, suggesting a dynamic turnover of this lipid. Fatty acid analysis indicated that most of the label was incorporated as palmitate. Lck was palmitoylated when expressed by transfection in COS cells. All of these protein tyrosine kinases were also detectably myristoylated in each of the systems tested. Experiments performed with mutants of Lck expressed by transfection in COS cells indicated that cysteines at positions 3 and 5 were both palmitoylation sites and that myristoylation was required for palmitoylation. To confirm that palmitoylation was occurring on cysteines in the N-terminal region of Fyn, site-directed mutagenesis was used to replace the cysteines at positions 3 and 6 with alanine. The resulting protein was not palmitoylated but was still myristoylated when expressed in COS cells. A glycine to alanine mutant at position 2 was also not palmitoylated, showing that myristoylation is a prerequisite for palmitoylation. Our data indicate that Src family members containing the N-terminal cysteine motif are indeed palmitoylated. By analogy with Ras, it is possible that palmitoylation may play an important role in the localization and function of Src family protein tyrosine kinases.

Amino Acid Sequence↗
PubMed1992

Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma.

Animals↗