Internalization Of Cell Surface Receptors Research Articles

Trafficking of the HIV Coreceptor CXCR4: ROLE OF ARRESTINS AND IDENTIFICATION OF RESIDUES IN THE C-TERMINAL TAIL THAT MEDIATE RECEPTOR INTERNALIZATION

The G protein-coupled chemokine receptor CXCR4 serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus. CXCR4 undergoes tonic internalization as well as internalization in response to stimulation with phorbol esters and ligand (SDF-1alpha). We investigated the trafficking of this receptor, and we attempted to define the residues of CXCR4 that were critical for receptor internalization. In both COS-1 and HEK-293 cells transiently overexpressing CXCR4, SDF-1alpha and phorbol esters (PMA) promoted rapid internalization of cell surface receptors as assessed by both enzyme-linked immunosorbent assay and immunofluorescence analysis. Expression of GRK2 and/or arrestins promoted modest additional CXCR4 internalization in response to both PMA and SDF. Both PMA- and SDF-mediated CXCR4 internalization was inhibited by coexpression of dominant negative mutants of dynamin-1 and arrestin-3. Arrestin was also recruited to the plasma membrane and appeared to colocalize with internalized receptors in response to SDF but not PMA. We then evaluated the ability of CXCR4 receptors containing mutations of serines and threonines, as well as a dileucine motif, within the C-terminal tail to be internalized and phosphorylated in response to either PMA or SDF-1alpha. This analysis showed that multiple residues within the CXCR4 C-terminal tail appear to mediate both PMA- and SDF-1alpha-mediated receptor internalization. The ability of coexpressed GRK2 and arrestins to promote internalization of the CXCR4 mutants revealed distinct differences between respective mutants and suggested that the integrity of the dileucine motif (Ile-328 and Leu-329) and serines 324, 325, 338, and 339 are critical for receptor internalization.

Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors.

Exposure of mammalian cells to ultraviolet (UV) light or high osmolarity strongly activates the c-Jun amino-terminal protein kinase (JNK) cascade, causing induction of many target genes. Exposure to UV light or osmotic shock induced clustering and internalization of cell surface receptors for epidermal growth factor (EGF), tumor necrosis factor (TNF), and interleukin-1 (IL-1). Activation of the EGF and TNF receptors was also detected biochemically. Whereas activation of each receptor alone resulted in modest activation of JNK, coadministration of EGF, IL-1, and TNF resulted in a strong synergistic response equal to that caused by exposure to osmotic shock or UV light. Inhibition of clustering or receptor down-regulation attenuated both the osmotic shock and UV responses. Physical stresses may perturb the cell surface or alter receptor conformation, thereby subverting signaling pathways normally used by growth factors and cytokines.

Agonist Regulation of α1B-Adrenergic Receptor Subcellular Distribution and Function

We have monitored agonist-induced alpha 1B-adrenergic receptor (alpha 1BAR) redistribution by immunocytochemical procedures in concert with functional measurements of agonist-elicited [3H]inositol phosphate (InsP) production in human embryonal kidney 293 cells stably expressing alpha 1BAR cDNA (HEK293/alpha 1B). Anti-peptide antibodies directed against the carboxyl-terminal decapeptide of the alpha 1BAR were prepared and shown to react specifically with alpha 1BAR on immunoblots and in situ in HEK293/alpha 1B transfectants. Treatment of HEK293/alpha 1B cells with norepinephrine (10 microM) results in a rapid (5-15 min) and striking internalization of cell surface receptor as visualized by confocal immunofluorescence microscopy. Receptor redistribution is sustained in the presence of agonist, rapidly reversed upon agonist removal, and prevented by the alpha 1 antagonist prazosin. Receptor internalizes to endosomes, as shown by colocalization with transferrin receptor, an endosomal marker. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (50 nM) causes receptor endocytosis similar to agonist; agonist-induced internalization is blocked by the PKC inhibitor staurosporine (0.5 microM). In parallel experiments, agonist-induced [3H]InsP production is abolished by phorbol 12-myristate 13-acetate but potentiated by staurosporine. Inhibition of receptor internalization with hypertonic sucrose attenuates agonist-induced [3H]InsP formation; this effect is reversed by concomitant inhibition of PKC with staurosporine. These results suggest that PKC-dependent phosphorylation occurring as a consequence of alpha 1AR stimulation induces receptor desensitization and internalization. Internalized receptor is reactivated and continuously recycled to the cell surface during agonist exposure.

The regulation of oxytocin receptor expression in human myometrial monolayer culture.

Clarification of the mechanism of oxytocin (OT)-induced contraction of the uterus seems to be essential for the elucidation of the mechanism of the initiation of labor. Although it has been suggested that estradiol (E) and progesterone (P) are involved in the expression of oxytocin receptors (OTRs), no consensus opinion has been formed on this topic. Thus we recently assessed the effects of E and P on OTR expression using cultures of human uterine cells and we examined the changes in the expressed OTRs following treatment of the cell with exogenous OT. The following results were obtained: 1. The total cellular concentration of OTRs as measured in the myometrial crude membrane fraction (total OTR concentration) showed an increase dependent on the E concentration and on the length of the incubation period in the presence of E. This increase following treatment with E (10(-7) M) was suppressed by simultaneous treatment with P in a concentration-dependent manner; being suppressed by 50% when the concentration of P was 2.7 x 10(-6) M (E/P ratio = 0.037). When 1 nM OT was added to the culture, the total OTR concentration did not change within the first 30 minutes of incubation, but decreased by about 70% twenty-four hours after addition of OT. 2. The concentration of OTRs at the cell surface (surface OTR concentration), as measured in myometrial cells that adhered to the culture plate, did not change with time when the cells were cultured at 4 degrees C in the presence of 1 nM OT. However, when cultured at 37 degrees C, the surface OTR concentration showed decreases dependent on the concentration of OT added and the time after the addition of OT. This change was observed within 60 minutes after the addition of OT to the culture. This decrease in surface OTR concentration was suppressed by concanavalin A (ConA), an inhibition of the internalization of cell surface receptors. These results indicate that in humans also, the expression of myometrial OTRs is regulated by change in the E/P ratio. The present study also revealed that OTRs, once expressed, soon disappear from the cell surface in the presence of exogenous OT (due to internalization of OTRs, i.e., dislocation of OTRs from the cell surface to the inside of the cells), and that prolonged exposure to OT even leads to the disappearance of intracellular OTRs. The present study thus suggests that the expression of human myometrial OTRs is regulated by E and P, and that an agonist-induced desensitization mechanism at the receptor level, similar to that reported for beta-adrenergic receptors, is also operating in this receptor.

Divergent conformational requirements for angiotensin II receptor internalization and signaling

Internalization is a general feature of plasma membrane receptors, including those for nutrients (such as LDL, transferrin), growth factors (insulin, EGF, FGF, etc.), and agonists that activate U protein-coupled responses (-adrenergic and muscarinic ligands, peptide hormones, etc.). The function of this process in delivering nutrients to the cells is well established, for example, via the LDL and the transferrin receptors. The internalization process also plays a key role in regulating the number of cellsurface receptors for hormones and growth factors, and has been proposed to be important for resensitization of /3-adrenergic and possibly other receptors [11. In addition, recent data indicate that the receptor internalization process might have a function in the signal generation process of growth factor and Ca2 mobilizing receptors [2—4]. Internalization of plasma-membrane receptors is preceded by accumulation of the receptors in specialized coated pit regions of the cell surface. Coated pits invaginate and pinch off to form coated vesicles that carry the receptor into the cell. The contents of the endocytic vesicles accumulate in endosomes, and the internalized receptors and their ligands either recycle to the cell surface or are targeted for lysosomal degradation. The processes responsible for internalization of cell-surface receptors can be divided into two major groups. Most nutrient receptors undergo constitutive internalization, whereas hormone and growth factor receptors that generate intracellular signals, undergo endocytosis that is activated by their specific ligands [1, 2, 5, 6]. The assembly unit of the polygonal lattice on the surface of coated pits and vesicles is the clathrin triskeleton. Clathrin tnskeletons are three-legged structures consisting of three heavy chains and three tightly associated light chains [7]. Most available data indicate that the clathnin structure binds to the membrane via one of the major classes of coat proteins recently named adaptor proteins (APs) [7]. Two structurally related classes of APs are present in most cell types. Immunofluorescence studies revealed that the APi complex colocalizes with the Golgi-associated coated pits, while the AP2 is found in plasma membrane-associated

Direct Evidence for Ligand-Induced Internalization of the Yeast α-Factor Pheromone Receptor

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before alpha-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. The median transit time in both assays was approximately 8 min. The ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofractionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation was blocked in a pep4 mutant that is deficient for vacuolar proteases. The carboxy-terminal domain of the receptor that is required for ligand internalization was also found to be essential for endocytosis of the receptor. A receptor mutant, ste2-L236H, which is defective for pheromone response but capable of ligand internalization, was found to be proficient for receptor endocytosis. Hence, separate structural features of the receptor appear to specify its signal transduction and internalization activities.

Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor.

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before alpha-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. The median transit time in both assays was approximately 8 min. The ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofractionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation was blocked in a pep4 mutant that is deficient for vacuolar proteases. The carboxy-terminal domain of the receptor that is required for ligand internalization was also found to be essential for endocytosis of the receptor. A receptor mutant, ste2-L236H, which is defective for pheromone response but capable of ligand internalization, was found to be proficient for receptor endocytosis. Hence, separate structural features of the receptor appear to specify its signal transduction and internalization activities.

Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit.

The juxtamembrane region of the insulin receptor (IR) beta-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXY [corrected] is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IR delta 960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXY [corrected], were deleted. Although the in vivo autophosphorylation of IRF960 and IR delta 960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR delta 960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IR delta 960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IR delta 960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXY [corrected] may play a general role in directing the internalization of cell surface receptors.