Growth control of hybridoma cells with an artificially induced EpoR-gp130 heterodimer

IL-6 has been known to modulate the growth of many hybridoma cells and also promote resultant antibody productivity. However, IL-6 is so expensive that the use of IL-6-dependent hybridomas for industrial antibody production is not practical. In this study, we aimed at designing antibody/gp130 and antibody/EpoR chimeras which could tightly control cell growth in response to more affordable cognate antigen. Retroviral vectors encoding V_H or V_L region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2/transmembrane domains of erythropoietin receptor (EpoR) and cytoplasmic domains of either EpoR or gp130, were constructed, and a homodimeric or a heterodimeric pair of chimeric receptor combinations (V_H-gp130 and V_L-gp130 or V_H-gp130 and V_L-EpoR) were expressed in an IL-6-dependent hybridoma 7TD1. The chimeric receptor-derived growth signal was observed in both combinations, while some residual growth signal was observed in the absence of HEL. To reduce interchain interaction between the two receptor chains, we introduced mutations to the transmembrane domain of both chimera combinations. Consequently, the heterodimeric combination of V_H-gp130 and V_L-EpoR showed clear HEL-dependent cell growth, while the homodimeric combination of V_H-gp130 and V_L-gp130 showed reduced cell growth in the absence of HEL. This is the first report that an EpoR-gp130 cytoplasmic domain heterodimer could transduce a growth signal in hybridoma cells, indicating tight and economical growth control of hybridoma cells via our chimeric receptors.     

Functionally essential cytoplasmic domain of the erythropoietin receptor.

The cytoplasmic domains of the erythropoietin receptor essential for signal transduction were identified by assessing a series of truncated and deletional mutant receptors. A 91-amino acid region proximal to the transmembrane domain was required for growth signaling. In this region, residues between 353Pro and 362His and between 278Gln and 308Leu appeared to constitute the essential cytoplasmic domains. These two domains contain the conserved amino acids common in the cytokine receptor superfamily, which indicates that these domains in the cytoplasmic regions of the erythropoietin receptor may be important for interaction with common signal transducers or protein tyrosine kinases.     

The role of syndecan cytoplasmic domain in basic fibroblast growth factor-dependent signal transduction.

To determine the role played by syndecan-4 cytoplasmic domain in the mediation of basic fibroblast growth factor (bFGF) signaling, immortalized human cells (ECV) were used to generate cell lines expressing constructs encoding full-length sequences for syndecan-4 (S4), syndecan-1 (S1), glypican-1 (G1), or chimeric proteins consisting of the ectoplasmic domain of glypican-1 linked to the transmembrane/cytoplasmic domain of syndecan-4 (G1-S4c) and the ectoplasmic domain of syndecan-4 linked to the glypican-1 glycosylphosphatidylinositol (GPI) anchor sequence (S4-GPI). Vector-transduced cells (VC) were used as controls. Expression of all these proteoglycans (except for the vector control) significantly increased cell-associated heparan sulfate mass and the number of low affinity bFGF-binding sites. However, in low serum medium, the addition of bFGF stimulated growth and migration of cells expressing S4 and G1-S4c constructs but not G1, S1, S4-GPI, or VC cells. Similar results were obtained using Matrigel growth assays. Mutations of heparan sulfate attachment sites on S4 construct abolished syndecan-4-dependent augmentation of bFGF responses. We conclude that cytoplasmic tail of syndecan-4 plays an important role in bFGF-mediated signal transduction.     

Identification of an essential region for growth signal transduction in the cytoplasmic domain of the human interleukin-4 receptor.

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system by regulating proliferation and differentiation of a wide variety of lymphoid and myeloid cells. These biological effects are manifested via binding of IL-4 to specific membrane-associated high affinity receptors. While the IL-4 receptor (IL-4R) cDNA expresses high affinity binding sites when transfected in COS7 cells, its intracellular domain lacks consensus motifs for known signal transducing molecules such as a tyrosine kinase. In this study, we use a DNA deletion approach to explore the mechanism of signal transduction utilized by the human IL-4R cDNA expressed in a murine pro-B cell line, Ba/F3 cells. Using this system, we have identified the critical region of the cytoplasmic domain of human IL-4R for human IL-4-induced transduction of a growth signal in these cells. Our data indicate that the critical region for signal transduction is located between amino acid residues 433-473 numbering from the carboxyl terminus. This region is highly conserved between mouse and human IL-4R but lacks homology with other cytokine receptors. Our studies additionally demonstrate that the cytoplasmic domain is not essential for forming high affinity IL-4-binding sites nor for ligand internalization.     

Positive charges in the cytoplasmic domain of Escherichia coli leader peptidase prevent an apolar domain from functioning as a signal.

Leader peptidase, an integral transmembrane protein of Escherichia coli, requires two apolar topogenic elements for its membrane assembly: a 'hydrophobic helper' and an internal signal. The highly basic cytoplasmic region between these domains is a translocation poison sequence, which we have shown blocks the function of a preceding signal sequence. We have used oligonucleotide-directed mutagenesis to remove positively charged residues within this polar domain to determine if it is the basic character in this region that has the negative effect on translocation. Our results show that mutations that remove two or more of the positively charged residues within the polar region no longer block membrane assembly of leader peptidase. In addition, when the translocation poison domain (residues 30-52) is replaced with six lysine residues, the preceding apolar domain cannot function as an export signal, whereas it can with six glutamic acids. Thus, positively charged residues within membrane proteins may have a major role in determining the function of hydrophobic domains in membrane assembly.     

Self assembly of the transmembrane domain promotes signal transduction through the erythropoietin receptor

Hematopoietic cytokine receptors, such as the erythropoietin receptor (EpoR), are single membrane-spanning proteins. Signal transduction through EpoR is crucial for the formation of mature erythrocytes. Structural evidence shows that in the unliganded form EpoR exists as a preformed homodimer in an open scissor-like conformation precluding the activation of signaling. In contrast to the extracellular domain of the growth hormone receptor (GHR), the structure of the agonist-bound EpoR extracellular region shows only minimal contacts between the membrane-proximal regions. This evidence suggests that the domains facilitating receptor dimerization may differ between cytokine receptors. We show that the EpoR transmembrane domain (TM) has a strong potential to self interact in a bacterial reporter system. Abolishing self assembly of the EpoR TM by a double point mutation (Leu 240-Leu 241 mutated to Gly-Pro) impairs signal transduction by EpoR in hematopoietic cells and the formation of erythroid colonies upon reconstitution in erythroid progenitor cells from EpoR(-/-) mice. Interestingly, inhibiting TM self assembly in the constitutively active mutant EpoR R129C abrogates formation of disulfide-linked receptor homodimers and consequently results in the loss of ligand-independent signal transduction. Thus, efficient signal transduction through EpoR and possibly other preformed receptor oligomers may be determined by the dynamics of TM self assembly.     

Autophosphorylation of the insulin-like growth factor I receptor cytoplasmic domain

The cytoplasmic domain of the beta subunit of the insulin-like growth factor I receptor (amino acids 936-1337) was overexpressed in Sf9 insect cells using a baculovirus expression system, and the 6-His tagged receptor was purified by metal-affinity chromatography. Autophosphorylation of the receptor was concentration dependent, consistent with a trans phosphorylation mechanism. Phosphoamino acid analysis of the autophosphorylated receptor showed predominantly phosphotyrosine, but phosphoserine and phosphothreonine were also present. However, when the receptor was further purified by gel filtration on Sephadex G-100 and then autophosphorylated, phosphoamino acid analysis showed only phosphotyrosine. We conclude that the IGF-I receptor tyrosine kinase is not a dual-specificity kinase and that autophosphorylation of the beta subunit is by a trans mechanism.     

The cytoplasmic tyrosine motifs in full-length glycoprotein 130 have different roles in IL-6 signal transduction

The function of the signal-transducing receptor subunit glycoprotein 130 (gp130) in the IL-6-receptor complex has previously been studied using carboxyl-terminal deletion mutants or a truncated molecule of approximately 60 membrane-proximal amino acids (containing box 1 and box 2) linked to the individual gp130 tyrosine motifs. However, the redundancy of the tyrosine motifs within the cytoplasmic part of gp130 has been neglected. Here we describe the analysis of the function of the individual cytoplasmic tyrosine residues of gp130 in the context of the full-length receptor protein in IL-6 signaling as measured by STAT activation, acute phase protein induction, and stimulation of proliferation. Add-back receptor mutants containing only one cytoplasmic tyrosine have been generated and tested for their efficiency in IL-6 signal transduction. Our studies revealed that tyrosine motifs which have been described to recruit STAT proteins are not equivalent with respect to their potential to activate STAT factors and acute phase protein gene promoters: the two distal tyrosines, Tyr905 and Tyr915, of gp130 were more potent than Tyr767 and Tyr814. Surprisingly, Tyr905 and Tyr915 mediate acute phase protein gene promoter activation stronger than the wild-type receptor containing all six cytoplasmic tyrosine residues. In contrast, Ba/F3 cells stably transfected with add-back receptors containing Tyr767 or Tyr905 were more sensitive to IL-6-induced proliferation than cells expressing the other add-back receptor mutants. Thus, the tyrosine residues in the cytoplasmic part of gp130 were found to contribute differentially to IL-6 signal transduction in the full- length gp130 protein.     

Effect of an artificially induced drought on leaf peroxidase activity, mineral nutrition and growth of sugar maple

The effect of an artificially induced drought was investigated in an 80-year-old sugar maple (Acer saccharum Marsh.) stand in southern Quebec. We hypothesized that leaf peroxidase activity would increase, and that leaf nutrient deficiencies typical of declining trees (K, Mg) would develop during the year following the imposition of the drought. An impermeable tarpaulin was placed on the ground at the base of the trees during the summer of 1991. Foliage and soils were sampled periodically during the summers of 1991 and 1992. The treated trees showed decreased leaf water potential and increased peroxidase activity over most of the 1991 growing season. Diameter growth was significantly reduced in 1992. Soil NO ₃ ^- and K⁺ were elevated while Mn²⁺ was reduced under the tarpaulin treatment at various times during the 1991 growing season. Soil NO ₃ ^- , NH ₄ ⁺ , K⁺ and Mg²⁺ were elevated in the treated plots at various times during the 1992 growing season even though there was a lack of effect on foliar nutrient concentrations over the same period. Drought conditions may have reduced water and nutrient uptake without significantly affecting leaf nutrient status.     

Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation.

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.     

Treatment of artificially induced storage disease with lysosomotropic microparticles

An artificial storage disease has been induced in mice by intravenously injecting 14C-labelled crosslinked dextran. The dextran was essentially taken up in the spleen and liver by cells belonging to the reticuloendothelial system and stored there in the lysosomes. Soluble dextranase, when given intravenously, had no effect on the stored dextran as judged by the radioactivity content of the tissues. However, when the dextranase was immobilized in microparticles of polyacryldextran and injected intravenously, the clearance of the radiolabelled dextran was significantly catalyzed. The lysosomotropic microparticles carried the enzyme to the lysosomes, where the microparticles were destroyed by the dextranase, which thereafter digested the stored 14C-dextran. The results illustrate the use of "self-destroying" microparticles as a lysosome-directed carrier of enzymes.     

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.     

In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.     

An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor

The polymeric immunoglobulin receptor is normally delivered from the Golgi to the basolateral surface of epithelial cells and then transports polymeric IgA and IgM to the apical surface. We now report that a 14 residue segment of the 103 amino acid cytoplasmic domain, proximal to the plasma membrane, directs the receptor to the basolateral surface. A mutant receptor lacking these 14 amino acids is sorted directly to the apical surface from the Golgi. Furthermore, this sequence is sufficient to redirect an apical membrane protein, placental alkaline phosphatase, to the basolateral plasma membrane. We conclude that this sequence contains an autonomous signal, which specifies sorting from the Golgi to the basolateral surface, a process previously postulated to occur by default.     

A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.     

Generation of recombinant cytoplasmic domain of epidermal growth factor receptor with intrinsic protein tyrosine kinase activity

We have generated a recombinant baculovirus using the high expression vector pVL941 containing the complementary DNA encoding the intracellular domain of the human epidermal growth factor receptor (EGFR-IC). Upon infection of Spodoptera frugiperda insect cells, protein tyrosine kinase-active EGFR-IC was produced. The expressed protein has a molecular weight of 61,000 and is specifically recognized by antibodies directed against peptides representing different regions of human EGFR-IC. Upon sonication of infected cells, EGFR-IC was detected in both the soluble and insoluble fractions of the cell lysate. About 20-50% of the expressed EGFR-IC was soluble. Metabolic labeling and protein analyses showed that EGFR-IC comprised 7% of newly synthesized proteins in the cytoplasmic lysate and 0.1-0.2% of the total soluble protein. We have used a three-step purification procedure (fast-Q-Sepharose and phenyl-Sepharose column chromatographies and 30% ammonium sulfate precipitation) to purify EGFR-IC to 85% purity with 15-20% recovery from the initial soluble lysate. A yield of 3-4 mg of purified EGFR-IC has been consistently produced from 20 roller bottles with 2-4 x 10(8) infected cells/bottle. The tyrosine kinase activity was retained through purification. The enzyme demonstrated much higher autophosphorylation activity in the presence of Mn2+ than Mg2+. Phosphopeptide mapping revealed the same autophosphorylation sites utilized by EGFR-IC as those identified in wild-type EGFR. EGFR-IC-catalyzed phosphorylation of either a synthetic peptide representing the major autophosphorylation site or angiotensin II showed that the baculovirus-expressed EGFR-IC exhibits similar enzymatic kinetic characteristics to the intact activated EGFR kinase.     

The structure of the unliganded extracellular domain of the interleukin-6 signal transducer gp130 in solution

Interleukin-6 (IL-6) plays an important role in immune responses and signals via two different pathways. When IL-6 binds to its non-signalling membrane-bound receptor (IL-6R), a non-covalent dimer of the ubiquitous interleukin-6 signal transducer gp130 is recruited to initiate intracellular signalling cascades. This so-called classical signalling pathway is restricted to cells expressing the membrane-bound IL-6R, such as hepatocytes and certain leukocytes. In addition, an alternative trans-signalling pathway uses soluble forms of IL-6R (sIL-6R) in complex with IL-6 to activate cells expressing gp130, but not membrane-bound IL-6R. In both cases, a tetrameric or hexameric signalling complex consisting of two gp130 molecules and one or two molecules each of IL-6 and (s)IL-6R is formed. The structure of the hexameric complex of the ligand-binding domains of gp130 (D1-D3) with IL-6 and sIL-6R has been solved by X-ray crystallography as well as the full-length extracellular part of gp130 (D1-D6) as a monomer. Since gp130 exists as a preformed dimer on the cell surface, we used a sgp130Fc fusion protein - consisting of two extracellular gp130 regions (D1-D6) dimerised by an IgG1-Fc part - to study the structure of unliganded gp130 extracellular domains in solution by small-angle X-ray scattering (SAXS). The SAXS data indicated that sgp130Fc forms a rigid molecule in solution. The low resolution structural model reveals an elongated assembly with an Fc base and two gp130 arms, whereby the orientation of the ligand-binding domains D1-D3 with respect to the membrane-proximal domains D4-D6 differs from that in the crystallographic monomer. Functional implications of these findings are discussed.     

Binding of madindoline A to the extracellular domain of gp130

Elevated levels of IL-6 and IL-11 are associated with multiple myeloma, rheumatoid arthritis, hypercalcemia, cancer cachexia, and Castleman's disease. Madindoline A (MadA), isolated from Streptomyces nitrosporeus K93-0711, specifically inhibits the growth of IL-6- and IL-11-dependent cell lines, most likely by interfering with the homodimerization of gp130. This raises the possibility that MadA can be used as a model compound for the development of novel chemotherapeutic agents. In this report, we demonstrate that the binding of MadA to gp130 is specific and noncovalent, and displays a relatively low affinity. Furthermore, we show that the tricyclic 3a-hydroxytetrahydrofuro[2,3-b]indole (HFI) moiety of MadA alone is not sufficient for binding. Matrix-bound MadA precipitates a protein composed of the extracellular domain of gp130 fused to the Fc region of the immunoglobulin heavy chain. Binding is inhibited in a dose-dependent manner by preincubation with free MadA. The K(D) for binding of MadA to gp130 is 288 microM, as determined by surface plasmon resonance (SPR)-based biosensor analysis. The HFI portion of MadA does not bind to gp130 in either affinity precipitation or SPR analyses. Finally, MadA, but not the HFI portion, inhibits IL-6-dependent Stat3 tyrosine phosphorylation in HepG2 cells.     

Molecular cloning and expression of an IL-6 signal transducer, gp130

Interleukin-6 (IL-6) signal is transduced through a membrane glycoprotein, gp130, which associates with IL-6 receptor (IL-6-R). A cDNA encoding human gp130 has been cloned, revealing that it consists of 918 amino acids with a single transmembrane domain. The extracellular region comprises six units of a fibronectin type III module, and part of this region of approximately 200 amino acids has features typical of a cytokine receptor family. A cDNA-expressed gp130 showed no binding property to IL-6 or several other cytokines. Although a transfectant with an IL-6-R cDNA expressed mainly low affinity IL-6 binding sites, an increase in high affinity binding sites was observed after cotransfection with a gp130 cDNA. This confirmed that a gp130 is involved in the formation of high affinity IL-6 binding sites. A cloned gp130 could associate with a complex of IL-6 and soluble IL-6-R and transduce the growth signal when expressed in a murine IL-3-dependent cell line.     

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.