Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants

Terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium, that are bound by a canonical DDXXD motif, as well as a putative second, seemingly less well conserved and understood (N/D)DXX(S/T)XXXE motif. Given the role of the Ser/Thr side chain hydroxyl group in ligating one of the three catalytically requisite divalent metal ions and the loss of catalytic activity upon substitution with Ala, it is surprising that Gly is frequently found in this ‘middle’ position of the putative second divalent metal binding motif in plant TPS. Herein we report mutational investigation of this discrepancy in a model plant diterpene cyclase, abietadiene synthase from Abies grandis (AgAS). Substitution of the corresponding Thr in AgAS with Ser or Gly decreased catalytic activity much less than substitution with Ala. We speculate that the ability of Gly to partially restore activity relative to Ala substitution for Ser/Thr stems from the associated reduction in steric volume enabling a water molecule to substitute for the hydroxyl group from Ser/Thr, potentially in a divalent metal ion coordination sphere. In any case, our results are consistent with the observed conservation pattern for this putative second divalent metal ion binding motif in plant TPS.     

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.     

Threonine 41 in beta-catenin serves as a key phosphorylation relay residue in beta-catenin degradation

Beta-catenin phosphorylation at serine 45 (Ser45), threonine 41 (Thr41), Ser37, and Ser33 is critical for beta-catenin degradation, and regulation of beta-catenin phosphorylation is a central part of the canonical Wnt signaling pathway. Beta-catenin mutations at Ser45, Thr41, Ser37, and Ser33 perturb beta-catenin degradation and are frequently found in cancers. It is established that Ser45 phosphorylation by casein kinase I (CKI) initiates phosphorylation at Thr41, Ser37, and Ser33 by glycogen synthase kinase 3 (GSK3) and that phosphorylated Ser37 and Ser33 are recognized by the F-box protein beta-TrCP, a component of a ubiquitin ligase complex that mediates beta-catenin degradation. While the roles of Ser45, Ser37, and Ser33 are well documented, the function of Thr41 remains less defined. Here we show that Thr41 strictly acts as a phosphorylation relay residue and that the Ser-X-X-X-Ser (X is any amino acid) motif is obligatory for beta-catenin phosphorylation by GSK3. Beta-catenin phosphorylation/degradation and its regulation by Wnt can occur normally in the absence of Thr41 as long as the Ser-X-X-X-Ser motif/spacing is preserved. These results suggest that Thr41 functions to bridge sequential phosphorylation from Ser45 to Ser37 and provide further insights into the discrete steps and logic in beta-catenin phosphorylation-degradation.     

Evidence for unique homologous peptide sequences around the glycosylated seryl and threonyl residues in polysialoglycoproteins isolated from the unfertilized eggs of the Pacific salmon Oncorhynchus keta

Structures of glycopeptides obtained by exhaustive Pronase digestion of high molecular weight (1.7 X 10(5)) salmon egg polysialoglycoprotein have been elucidated. Six principal glycopeptides isolated by gel chromatography and DEAE-Sephadex A-25 chromatography in the absence or presence of borate ion were analyzed for their carbohydrate and amino acid composition, as well as amino acid sequence, and found to be of two distinct types: glycotripeptides, Thr*-Ser*-Glu, and glycotetrapeptides, Thr*-Gly-Pro-Ser, where an asterisk indicates the amino acid residues to which either the Gal beta 1----3GalNAc or Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3GalNAc chain is attached. Their final yield corresponds to 64% of the original desialylated glycoprotein. In view of the simple amino acid composition of salmon egg polysialoglycoprotein (molar ratio Asp2Thr2Ser3Glu1Pro1Gly1Ala3) and the result of alkaline beta-elimination indicating three carbohydrate units linked to two of two threonine and one of three serine residues, a unique primary structure comprising repetitive sequences of the above two types of glycopeptides, which are interspersed by short nonglycosylated peptides consisting of alanine and aspartic acid, has been proposed for the core protein. The molecular secondary ion mass spectra of underivatized glycopeptides were used to obtain their structural information. The anomeric configuration of the proximal sugar-peptide linkages was proven to be alpha by proton nuclear magnetic resonance spectroscopy. This is the first systematic reported study of O-glycosidically linked glycopeptides by these instrumental methods.     

The dysfunctional LDL receptor in a monensin-resistant mutant of Chinese hamster ovary cells lacks selected O-linked oligosaccharides

The Chinese hamster ovary (CHO) cell line Monr31, which is resistant to the cytotoxic ionophore monensin, produces a receptor for the low density lipoprotein (LDL) that has a lowered binding affinity for LDL and is approximately 5 kDa smaller in size than the receptor from parental CHO cells. It has been proposed that the reduced size and affinity for LDL are associated with a reduced level of O-glycosylation of Ser/Thr residues in the receptor. To examine this possibility in more detail, both parental CHO and Monr31 cells were metabolically radiolabeled with [3H]glucosamine, and the labeled LDL receptors were purified by immunoprecipitation and identified by SDS-PAGE-fluorography. The Ser/Thr-linked oligosaccharides in the receptors from both parental CHO and Monr31 cells are mono- and desialylated species having the common core structure Gal beta 1-3GalNAc. The receptor from Monr31 cells, however, contains about one-third fewer Ser/Thr-linked oligosaccharides than the receptor from parental CHO cells. Analysis of the glycopeptides derived from the Monr31 cell LDL receptors indicates that they contain Ser/Thr-linked oligosaccharides only in the clustered domain and are missing Ser/Thr-linked oligosaccharides in the unclustered regions of the protein. Additionally, analysis of a human LDL receptor lacking the domain for attachment of the clustered Ser/Thr-linked oligosaccharides and expressed in both parental CHO and Monr31 cells indicated that the truncated human receptor from Monr31 cells is devoid of Ser/Thr-linked oligosaccharides. In contrast, the truncated human receptor produced by parental CHO cells contains Ser/Thr-linked oligosaccharides contributing approximately 5 kDa to its apparent size. Collectively, these results demonstrate that the LDL receptor produced by the Monr31 cells contains Ser/Thr-linked oligosaccharides in the clustered domain but is missing Ser/Thr-linked oligosaccharides in the unclustered, NH2-terminal domains of the receptor.     

Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture of medium of Diplococcus pneumoniae.

The culture medium of Diplococcus pneumoniae contains enzymic activity that cleaves Galbeta1 leads to 3GalNAc from desialized human erythrocyte membrane glycoprotein. The enzyme was purified 180-fold by ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and DEAE A-25 Sephadex chromatography. The purified enzyme liberates Galbeta1 leads to 3GalNAc from glycopeptides and glycoproteins with Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr moieties. The optimum pH of this enzyme is 6.0. Using glycopeptides obtained by trypsin digestion of human erythrocyte membrane glycoprotein as a substrate, a Km of 0.20 mM (on the basis of the amount of Galbeta1 leads to 3GalNAc residues) was obtained. So far, the enzyme appears to have a strict specificity for Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr structures, because no oligosaccharides larger than trisaccharides were liberated from porcine submaxillary mucin.     

A common peptide stretch among enzymes localized to the Golgi apparatus: structural similarity of Golgi-associated glycosyltransferases

A common peptide motif has been discovered among a series of Golgi-localized glycosyltransferases. The peptide stretch, (Ser/Thr)-X-(Glu/Gln)-(Arg/Lys), always occurs near a hydrophobic domain close to the N-terminus of these enzymes which is believed to anchor them to the membrane lipid bilayer (Paulson and Colley, J. Biol. Chem., 264, 17615-17618, 1989). The finding that this similar peptide motif is not associated with catalytic activity of these enzymes, and its presence near the hydrophobic domain suggest that the stretch may be involved in localization of these enzymes to the Golgi apparatus.     

Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A

The human red blood cell sialoglycoprotein, glycophorin A (GpA),contains a ‘mucin-like’ extensively O-glycosylatedextracellular domain which carries the MN blood group antigens.We have revised the sites of O-glyccsylation in the extracellulardomain of GpA by automated solid-phase Edman degradation, whichallowed positive identification and quantitation of O-glycosylatedSer and Thr residues, as well as the single N-glycosylationsite. One N-linked and 16 O-linked sites were identified. Carbohydratewas absent on Ser 1, Ser14, Ser15, Ser23, Thr28 and Thr58 inGpA. We propose that the glycosyltransferases present in erythrocytesrecognize specific flanking sequences around potential O-glycosylationsites. All 16 O-glycosylation sites are explained on the basisof four motifs. Three motifs are associated with Thr-glycosylation:Xaa—Pro—Xaa—Xaa where at least one Xaa = Thr;Thr—Xaa—Xaa—Xaa where at least one Xaa = Thr;Xaa—Xaa—Thr—Xaa where at least one X = Argor Lys. The fourth motif is associated with Ser-glycosylation:Ser—Xaa—Xaa—Xaa where at least one Xaa = Ser.These simple rules explain the glycosylation (or lack of it)on 21 of 22 Ser/Thr in the extracellular domain of GpA. glycophorin A O-glycosylation motif solid-phase Edman degradation     

Isolation and structural characterization of sialic-acid-containing glycopeptides of the O-glycosidic type from the urine of two patients with an hereditary deficiency in alpha-N-acetylgalactosaminidase activity

Glycopeptides have been isolated from the urine of two patients, aged 5 and 6, with a new lysosomal storage disease characterized by a deficiency in alpha-N-acetylgalactosaminidase activity. Isolation of these glycopeptides was achieved using gel filtration and ion-exchange chromatography. Structural determination was done using one- and two-dimensional 500 MHz 1H-NMR spectroscopy and FAB mass spectrometry of native and derivatized glycopeptides. The following structures were inferred as being present: Glycopeptide A (up to 140 mg/l urine) (1)-(3) Neu5Ac alpha 2-3Gal beta 1-3 (Neu5Ac alpha 2-6)GalNAc alpha 1-R A1: R = Ser A2: R = Thr A3: R = Thr-Pro Glycopeptide B (up to 80 mg/l urine) (4)-(6) Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6 (Neu5Ac alpha 2-3-Gal beta 1-3) GalNAc alpha 1-R B1: R = Ser B2:R = Thr B3: R = Thr-Pro     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.     

Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites

It has recently been shown that caldesmon from non-muscle (Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172) and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding to F-actin and Ca(2+)-calmodulin. In this study, we have identified five phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667, Thr673, Thr696, and Ser702. All the sites bear some resemblance to the S(T)-P-X-X motif recognized by p34cdc2. The preferred site of phosphorylation at Thr673 accounts for about 40% of the total phosphorylation. Four of the sites occur in two pairs of closely spaced sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in each pair inhibits strongly the phosphorylation of the second site in the same pair, presumably due to the close proximity of the two sites. Similar negative cooperativity in phosphorylation of Ser667 and Thr673 was observed using a 22-residue synthetic peptide containing the two sites. Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90% of the total level of phosphorylation and these sites are located within the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to bind actin and Ca(2+)-calmodulin.     

Characterization of a bacterial beta-1,3-galactosyltransferase with application in the synthesis of tumor-associated T-antigen mimics

T-Antigen (Gal-beta1,3-GalNAc-alpha-O-Ser/Thr) is an important precursor of mucin-type O-glycans. T-Antigen is found to be closely associated with cancer progression and metastasis and has been used to develop carbohydrate-based anticancer vaccines. Enzymatic synthesis of T-antigen disaccharides have relied on the use of beta-1,3-galactosyltransferases recently cloned and characterized from several eukaryotic organisms. However, its application is limited by the difficulty of obtaining homogeneous enzymes and the strict substrate specificity of enzymes. Recently, a number of bacteria have been found to express carbohydrate structures that mimic host glycans. The corresponding glycosyltransferases have been exploited in the facile synthesis of a number of clinically important glycoconjugate mimics. In this study, we biochemically characterized a bacterial beta-1,3-galactosyltransferase (WbiP) from Escherichia coli O127, which expresses a T-antigen mimic in the lipopolysaccharide (LPS) structure. Substrate study showed that WbiP could readily glycosylate a series of N-acetylgalactosamine (GalNAc) analogues with alpha-substitutions at the reducing end, including glycosylated Ser and Thr (GalNAc-alpha-O-Ser/Thr), which illustrates the use of WbiP for the facile synthesis of T-antigens. Alignment of a group of putative bacterial beta-1,3-galactosyltransferases revealed the presence of two conserved DXD motifs, possibly suggesting a different functional role of each motif. Site-directed mutagenesis, enzyme kinetics as well as UDP-bead binding assays were carried out to investigate the role of each DXD motif in WbiP. The results suggest that 88DSD90 is critical in the binding of sugar donor UDP-Gal, whereas 174DYD176 may participate in the binding of the sugar acceptor. This study expands the scope of using bacterial glycosyltransferases as tools for in vitro synthesis of glycoconjugate mimics with clinical significance.     

The role of the Ser/Thr cluster in the phosphorylation of PPPSP motifs in Wnt coreceptors

Wnt/β-catenin signaling controls a variety of cellular processes, including cell growth, oncogenesis, and development. Upon Wnt stimulation, the intracellular region of the coreceptor, LRP6 or 5, is phosphorylated by the membrane-recruited GSK3β and CK1. The cytoplasmic domain of LRP6/5 contains one Ser/Thr cluster and the PPPSP motifs, both of which are essential for propagation of the signal. While the phosphorylated PPPSP motifs are known to directly inhibit GSK3β, the biochemical role of the phosphorylated Ser/Thr cluster remains to be elucidated. Herein, we reveal that the Ser/Thr cluster plays an important role in the phosphorylation of the PPPSP motif. Interestingly, we observe that GSK3β activity on the PPPSP motif requires a high ATP concentration, close to that of the physiological condition. Taken together, these data suggest that the phosphorylated Ser/Thr cluster serves as a docking site for GSK3β to promote the phosphorylation of the PPPSP motif. Our results provide insight into the molecular mechanism for the initial events of the Wnt/β-catenin signaling.     

Conformational consequences of protein glycosylation: preparation of O-mannosyl serine and threonine building blocks, and their incorporation into glycopeptide sequences derived from alpha-dystroglycan

With the goal to investigate the structural impact of O-mannosyl glycosylation on alpha-dystroglycan, a glycoprotein that has an important role in the extracellular organization of muscle, glycopeptides derived from its mucin-like sequence have been prepared by solid-phase peptide synthesis. Two approaches have been explored to obtain needed mannosylated serine and threonine building blocks. With the alpha-carboxyl group unprotected, and with tetraaceto-1-fluoro-alpha-D-mannose as the sugar donor, the desired alpha-O-mannosyl-Fmoc-Ser/Thr formed, along with mannosyl ester isomers and the species with mannose attached to both hydroxyl and carboxyl functions. Relevant mechanistic questions and stability issues were elucidated. Alternatively, building blocks were made with the alpha-carboxyl protected/activated as the pentafluorophenyl (Pfp) ester. Glycopeptides synthesized herein contained 5-9 residues, and featured one, two, and four consecutive Ser and/or Thr residues O-glycosylated with mannose. Circular dichroism (CD) spectra for Man-containing glycopeptides recorded in water show them to be not well ordered. For one of the alpha-dystroglycan-derived sequences, the comparative conformational consequences of glycosylation by either Man or GalNAc have been examined by CD and NMR, with both methods showing a more organized structure when GalNAc is present.     

Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation

Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the alphaC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered alphaC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2.     

The ST Pinch: A side chain-to-side chain hydrogen-bonded motif

The ST Pinch is a 12-membered hydrogen-bonded motif (Ser/Thr-Xaa-Ser/Thr) involving the side chain oxygen atoms of two Ser/Thr residues. We identified the ST Pinch in 104 proteins in a database containing high-resolution crystal structures. Conformational analysis of the ST Pinch in these proteins points to specific preferences for the Xaa residue and a high propensity of this residue to adopt positive φ angles. Our results suggest that this motif serves as a linker of secondary structural elements within proteins and is a new addition to the existing list of short hydrogen bond-stabilized motifs in proteins.     

Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases

The TOR protein is a phosphoinositide kinase-related kinase widely conserved among eukaryotes. Fission yeast tor1 encodes an ortholog of TOR, which is required for sexual development and growth under stressed conditions. We isolated gad8, which encodes a Ser/Thr kinase of the AGC family, as a high-copy suppressor of the sterility of a tor1 mutant. Disruption of gad8 caused phenotypes similar to those of tor1 disruption. Gad8p was less phosphorylated and its kinase activity was undetectable in tor1Delta cells. Three amino acid residues corresponding to conserved phosphorylation sites in the AGC family kinases, namely Thr387 in the activation loop, Ser527 in the turn motif and Ser546 in the hydrophobic motif, were important for the kinase activity of Gad8p. Tor1p was responsible for the phosphorylation of Ser527 and Ser546, whereas Ksg1p, a PDK1-like kinase, appeared to phosphorylate Thr387 directly. Altogether, Tor1p, Ksg1p and Gad8p appear to constitute a signaling module for sexual development and growth under stressed conditions in fission yeast, which resembles the mTOR-PDK1-S6K1 system in mammals and may represent a basic signaling module ubiquitous in eukaryotes.     

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide.     

Phosphorylation of Ser363, Thr370, and Ser375 residues within the carboxyl tail differentially regulates mu-opioid receptor internalization

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between mu-opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. Among those 12 Ser/Thr residues, Ser(363), Thr(370), and Ser(375) have been identified as phosphorylation sites. In the absence of the agonist, a basal phosphorylation of Ser(363) and Thr(370) was observed, whereas [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr(370) and Ser(375) residues. Furthermore, the role of these phosphorylation sites in regulating the internalization of MOR was investigated. The mutation of Ser(375) to Ala reduced the rate and extent of receptor internalization, whereas mutation of Ser(363) and Thr(370) to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though mu-receptors and delta-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.