Biomedical subjects

R W Hipkin

Publications and source records attributed to R W Hipkin.

At least 19 recordsLinked to original sources

PubMed2001

Evaluation of signal transduction pathways in chemoattractant-induced human monocyte chemotaxis.

The intracellular signaling pathways involved in human monocyte chemotaxis toward a variety of chemoattractant molecules were evaluated using selected pharmacological agents. Neither phosphatidylinositol-3-kinase (P13K) or extracellular signal-regulated kinase (ERK) activity were required for monocyte migration toward monocyte chemoattractant protein-1 (MCP-1), RANTES (Regulated on Activation, Normal T cell Expressed and Secreted), macrophage inflammatory protein-1alpha (MIP-1alpha) or formyl-Met-Leu-Phe (fMLP), since pretreatment with wortmannin or LY294002, or with PD098059, had no effect on the chemotactic response. Addition of forskolin and IBMX significantly attenuated chemotaxis to each of these chemoattractants and was reversed by co-treatment with Rp-cAMP, a competitive inhibitor of cAMP-dependent protein kinase A. Incubation with the protein kinase C (PKC) inhibitor GF109203X-HCl (GF109) did not affect monocyte migration, but pretreatment of monocytes with PMA significantly impaired the response to each of these chemotactic agents. Inhibition by PMA was reversed by co-treatment with GF109, implying that heterologous PKC activation is capable of desensitizing chemokine and fMLP-induced monocyte chemotaxis. These results help to define the signalling pathways involved in human monocyte chemotaxis and suggest pharmacological approaches to evaluating the cross-desensitization of chemoattractant-induced leukocyte migration.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Human interferon-inducible 10-kDa protein and human interferon-inducible T cell alpha chemoattractant are allotopic ligands for human CXCR3: differential binding to receptor states.

The human CXC chemokines IP-10 (10-kDa interferon-inducible protein), MIG (monokine induced by human interferon-gamma), and I-TAC (interferon-inducible T cell alpha chemoattractant) attract lymphocytes through activation of CXCR3. In the studies presented here, we examined interaction of these chemokines with human CXCR3 expressed in recombinant cells and human peripheral blood lymphocytes (PBL). IP-10, MIG, and I-TAC were agonists in stimulating [(35)S]GTP gamma S binding in recombinant cell and PBL membranes but had no effect in the absence of hCXCR3 expression. (125)I-IP-10 and (125)I-I-TAC bound hCXCR3 with high affinity, although the (125)I-I-TAC B(max) value in saturation bindings was 7- to 13-fold higher than that measured with (125)I-IP-10. Coincubation with unlabeled chemokines decreased (125)I-IP-10 binding with a single discernible affinity. However, with (125)I-I-TAC, competition with IP-10 or MIG was incomplete, and multiple binding affinities were evident. Moreover, in contrast to I-TAC, IP-10 and MIG binding IC(50) values did not increase predictably with increased (125)I-I-TAC concentration in competition bindings, suggesting that these chemokines are noncompetitive (i.e., allotopic) ligands. Uncoupling of hCXCR3 eliminated (125)I-IP-10 binding but only decreased (125)I-I-TAC binding 30 to 80%, indicating that unlike IP-10, I-TAC binds with high affinity to uncoupled (R) and coupled (R*) hCXCR3. To examine chemokine binding to R*, we tested the effect of anti-hCXCR3 antibody on I-TAC- and IP-10-stimulated [(35)S]GTP gamma S binding. The antibody attenuated [(35)S]GTP gamma S binding in response to IP-10 but not to I-TAC, suggesting that the two chemokines bind differently to R*. Moreover, increased occupancy of R* with a >75-fold increase in (125)I -IP-10 concentration did not increase the I-TAC binding IC(50) value, and I-TAC increased the dissociation rate of (125)I-IP-10. From these data, we conclude that the binding of IP-10 and I-TAC to the R* state of hCXCR3 is allotopic.

Animals↗

PubMed2001

Cloning and characterization of a novel human histamine receptor.

Histamine exerts its numerous physiological functions through interaction with G protein-coupled receptors. Three such receptors have been defined at both the pharmacological and molecular level, while pharmacological evidence hints at the existence of further subtypes. We report here the cloning and characterization of a fourth histamine receptor subtype. Initially discovered in an expressed-sequence tag database, the full coding sequence (SP9144) was subsequently identified in chromosome 18 genomic sequence. This virtual coding sequence exhibited highest homology to the H(3) histamine receptor and was used to generate a full-length clone by polymerase chain reaction (PCR). The distribution of mRNA encoding SP9144 was restricted to cells of the immune system as determined by quantitative PCR. HEK-293 cells transiently transfected with SP9144 and a chimeric G protein alpha-subunit (Galpha(q/i1,2)) exhibited increases in intracellular [Ca(2+)] in response to histamine but not other biogenic amines. SP9144-transfected cells exhibited saturable, specific, high-affinity binding of [(3)H]histamine, which was potently inhibited by H(3) receptor-selective compounds. The rank order and potency of these compounds at SP9144 differed from the rank order at the H(3) receptor. Although SP9144 apparently coupled to Galpha(i), HEK-293 cells stably transfected with SP9144 did not exhibit histamine-mediated inhibition of forskolin-stimulated cAMP levels. However, both [(35)S]GTPgammaS binding and phosphorylation of mitogen-activated protein kinase were stimulated by histamine via SP9144 activation. In both of these assays, SP9144 exhibited evidence of constitutive activation. Taken together, these data demonstrate that SP9144 is a unique, fourth histamine receptor subtype.

Amino Acid Sequence↗

PubMed2000

Protein kinase C activation stimulates the phosphorylation and internalization of the sst2A somatostatin receptor.

The sst2A receptor is expressed in the endocrine, gastrointestinal, and neuronal systems as well as in many hormone-sensitive tumors. This receptor is rapidly internalized and phosphorylated in growth hormone-R2 pituitary cells following somatostatin binding (Hipkin, R. W., Friedman, J., Clark, R. B., Eppler, C. M., and Schonbrunn, A. (1997) J. Biol. Chem. 272, 13869-13876). The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), also stimulates sst2A phosphorylation. Here we examine the mechanisms and consequences of PMA and agonist-induced sst2A phosphorylation. Like somatostatin, both PMA and bombesin increased sst2A receptor phosphorylation within 2 min. The PKC inhibitor GF109203X blocked PMA- and bombesin- stimulated sst2A phosphorylation, whereas stimulation by the somatostatin analog SMS 201-995 was unaffected. Agonist and PMA each stimulated phosphorylation in two receptor domains, the third intracellular loop and the C-terminal tail. Functionally, PMA dramatically increased the internalization of the sst2A receptor-ligand complex. This PMA stimulation was blocked by GF109203X, whereas basal internalization was unaffected. However, neither basal nor PMA-stimulated internalization was altered by pertussis toxin, whereas both were blocked by hypertonic sucrose. Therefore PKC activation and agonist binding stimulate sst2A phosphorylation by distinct mechanisms, and PKC potentiates internalization of the sst2A receptor via clathrin-coated pits. Thus, hormonal stimulation of PKC-coupled receptors may provide a mechanism for regulating the inhibitory actions of somatostatin in target tissue.

Animals↗

PubMed2000

Endocannabinoid 2-arachidonyl glycerol is a full agonist through human type 2 cannabinoid receptor: antagonism by anandamide.

The endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) bind to G protein-coupled central and peripheral cannabinoid receptors CB1 and CB2, respectively. Due to the relatively high expression of the CB2 isotype on peripheral immune cells, it has been hypothesized that this receptor mediates the immunosuppressive effects of cannabinoids. Unfortunately, there was a dearth of pharmacological studies with the endocannabinoids and human CB2 (hCB2). These studies compare and contrast the potency and efficacy of anandamide, 2-AG, and the synthetic cannabinoid HU210 at hCB2. Using [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) and radioligand bindings in insect Sf9-hCB2 membranes, we showed that both endocannabinoids bound hCB2 with similar affinity and that the cannabinoids acted as full agonists in stimulating [(35)S]GTPgammaS exchange, although 2-AG was 3-fold more potent than anandamide (EC(50) = 38.9 +/- 3.1 and 121 +/- 29 nM, respectively). In a mammalian expression system (Chinese hamster ovary-hCB2 cells), HU210 and 2-AG maximally inhibited forskolin-stimulated cAMP synthesis (IC(50) = 1.61 +/- 0.42 nM and 1.30 +/- 0.37 microM, respectively) although anandamide was ineffective. In Chinese hamster ovary-hCB2 membranes, HU210 and 2-AG were also full agonists in stimulating [(35)S]GTPgammaS binding (EC(50) = 1.96 +/- 0.35 and 122 +/- 17 nM, respectively), but anandamide was a weak partial agonist (EC(50) = 261 +/- 91 nM; 34 +/- 4% of maximum). Due to its low intrinsic activity, coincubation with anandamide effectively attenuated the functional activity of 2-AG at hCB2. Collectively, the data showed that both endocannabinoids bound hCB2 with similar affinity, but only 2-AG functioned as a full agonist. Moreover, the agonistic activity of 2-AG was attenuated by anandamide.

Animals↗

PubMed1999

Pituitary somatostatin receptor (sst)1-5 expression during rat development: age-dependent expression of sst2.

The capacity of the pituitary to suppress hormone secretion in response to somatostatin (SRIF) is markedly age dependent. Immature pituitaries are relatively resistant to SRIF effects, and increasing sensitivity to SRIF with advancing age is believed to cause characteristic developmental changes in pituitary hormone secretion in mammals. However, the cellular mechanism(s) underlying this developmental pattern of response to SRIF are not understood. Because somatostatin receptors (ssts) are critical mediators of SRIF's actions on target tissues, we investigated the expression of sst1, sst2, sst3, sst4, and sst5 messenger RNA (mRNA) in pituitaries of developing and mature rats. Animals were studied at embryonic day 19.5, and at postnatal days 2, 12, 30, 45, 70, and 1 yr; these ages correspond to major changes in circulating GH levels and pituitary responsiveness to SRIF. Pituitary levels of sst2 mRNA increased strikingly and progressively with advancing age after birth (F = 30.92, P < 0.0001). Compared with 2-day-old pituitaries, sst2 mRNA abundance rose 3.25-fold by 12 days of age and 6-fold by 70 days of age. Moreover, Western blot analysis indicated a marked increase in pituitary expression of sst2A protein with advancing age. By contrast, pituitary abundance of sst1, sst3, sst4, and sst5 mRNAs did not differ with age. To assess the role of endogenous SRIF in regulating perinatal sst2 gene expression, we also administered a well-characterized SRIF antiserum (or NSS as controls; 10 microl/10 g) sc daily from postnatal days 2 to 12 of life. Treatment with SRIF antiserum raised GH levels but did not alter pituitary sst2 mRNA abundance, compared with controls. Taken together, these data indicate that 1) the perinatal rat pituitary expresses the same complement of ssts as the adult pituitary; 2) expression of ssts is developmentally regulated in a highly subtype-specific manner; 3) pituitary sst2 mRNA and sst2A protein increase markedly and progressively with advancing age after birth; and 4) the perinatal rise in sst2 mRNA levels is unlikely to be regulated by endogenous SRIF. The finding of subtype-specific, developmentally determined sst expression indicates a novel and potentially fundamental mechanism of sst regulation, and suggests a molecular mechanism underlying developmental maturation in the capacity of the pituitary to respond to SRIF.

Aging↗

PubMed1999

Immunohistochemical detection of somatostatin sst2a receptors in the lymphatic, smooth muscular, and peripheral nervous systems of the human gastrointestinal tract: facts and artifacts.

The cellular distribution of the somatostatin sst2A receptor protein was investigated in the lymphatic, smooth muscular, and nervous components of the human gastrointestinal tract using subtype-specific antibody R2-88 for immunohistochemical staining of cryostat and formalin-fixed, paraffin-embedded tissue sections. Germinal centers of intestinal lymphatic follicles were immunostained, exhibiting a predominantly plasma membrane localization of the receptor. Similarly, nerve fibers and cells in the submucosal and myenteric plexus were stained for sst2A. Antibody preabsorption with 100 nmol/L antigen peptide abolished staining in all of these tissues, and immunohistochemical staining correlated with the labeling observed after receptor autoradiography using the sst2-preferring radioligand 125I-[Tyr3]octreotide. Cytoplasmic immunostaining was detected in gastrointestinal smooth muscle cells and was inhibited by antibody pre-absorption with antigen peptide. However, 125I-[Tyr3]octreotide autoradiography was negative, and Western blots showed no band at the usual 70-90 kDa location for sst2A. Instead, a band was observed at 205 kDa. This band comigrated with the rabbit myosin standard, which was also stained with R2-88, although antibody sensitivity for myosin was less than 0.002% of that for the sst2A receptor. Rigorous computer-based sequence analysis demonstrated the peptide sequence chosen for antibody production was unique. Moreover, standard sequence alignment protocols were unable to identify the sequences in myosin responsible for the observed reactivity with the R2-88 antiserum. The observed cross-reactivity emphasizes the need for extensive controls to prove the specificity of immunostaining for such low abundance proteins as receptors even when the peptide sequence chosen for antibody production is unique. This study demonstrates for the first time the presence of specific sst2A receptor protein by immunohistochemistry in the human gastrointestinal lymphatic and nervous components, but not in gastrointestinal circular and longitudinal smooth muscle.

Animals↗

PubMed1998

Desensitization of beta2-adrenergic receptors with mutations of the proposed G protein-coupled receptor kinase phosphorylation sites.

Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the beta2-adrenergic receptor (betaAR) was recently reported based on in vitro phosphorylation of recombinant receptor (Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796-13803). Phosphorylated residues identified for GRK2 were threonine 384 and serines 396, 401, and 407. GRK5 phosphorylated these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites altered desensitization, we have constructed betaARs in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants betaARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 microM epinephrine treatment of cells. The mutant and wild type (WT) receptors were all desensitized 89-94% after 5 min of 10 microM epinephrine stimulation and 96-98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant betaARs relative to the WT in the extent of 10 microM epinephrine-induced internalization (77-82% after 30 min). Epinephrine treatment for 1 min induced a rapid increase in the phosphorylation of the GRK5 and PKA- mutant betaARs as well as the WT. We conclude that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivo GRK-dependent desensitization of the betaAR.

Adenylyl Cyclases↗

PubMed1998

Immunohistochemical localization of somatostatin receptors sst2A in human tumors.

Human tumors frequently express somatostatin receptors. However, none of the receptor subtype proteins have been individually visualized in normal or neoplastic human tissues. Here, the distribution of the sst2A receptor was investigated using immunohistochemistry with the specific anti-peptide antibody R2-88 in 47 human tumors. All tumors selected for their abundance of sst2 mRNA and/or strong binding of the sst2-preferring ligand 125I-labeled Tyr3-octreotide were specifically immunostained with R2-88. Conversely, all tumors without somatostatin binding or expressing predominantly other somatostatin receptor subtype mRNAs (sst1 or sst3) were not specifically immunostained by R2-88. Specificity was shown in immunoblots, demonstrating receptor migration as a 70-kd broad band. In immunohistochemical and immunoblotting experiments, the abolition of staining after antibody blockade with antigen peptide was demonstrated. Immunostaining was identified in cryostat and in formalin-fixed, paraffin-embedded sections. Heat-induced epitope retrieval was necessary to visualize sst2A receptors in formalin-fixed sections. Moreover, because of occasional high nonspecific staining, the demonstration of complete abolition of immunostaining by treatment with antigen peptide was a prerequisite for the correct identification of sst2A-positive tumors. The sst2A receptors were clearly located at the membrane of the tumor cells. These results provide the first localization of a somatostatin receptor subtype in human tissues at the cellular level. The sst2A receptor identification and visualization in tumors with simple immunohistochemical methods in formalin-fixed, paraffin-embedded material will open new diagnostic opportunities for pathologists.

Animals↗

PubMed1998

The agonist-induced phosphorylation of the rat follitropin receptor maps to the first and third intracellular loops.

Previous results from this laboratory have shown that the rat FSH receptor (rFSHR) becomes phosphorylated on S/T residues upon stimulation of transfected cells with human (h)FSH and that a truncation of the C-terminal tail that removes 12 of the 25 intracellular S/T residues does not affect phosphorylation. Based on the results of phosphopeptide-mapping experiments we analyzed three new mutants. rFSHR-1L and rFSHR-3L were constructed by mutating the S/T residues in the first intracellular loop or the third intracellular loop, respectively. rFSHR-(3L+CT) was constructed by mutating all the S/T residues in the third loop as well as S624, the only C-terminal tail residue that was not previously eliminated as a potential phosphorylation site. All mutants were biologically active. The agonist-induced phosphorylation of rFSHR-3L and rFSHR-(3L+CT) were partially reduced, while that of rFSHR-1L was almost completely lost. The agonist-induced uncoupling of rFSHR-1L and rFSHR-3L are retarded to about the same extent, while the agonist-induced internalization is retarded only in rFSHR-1L. Four major conclusions can be made from the present studies: 1) the phosphorylated rFSHR is a common molecular intermediate in agonist-induced uncoupling and internalization; 2) agonist-induced phosphorylation of the rFSHR maps to the first and third intracellular loops; 3) the phosphorylation of the third intracellular loop facilitates agonist-induced uncoupling but is not necessary for agonist-induced internalization; 4) agonist-induced internalization is facilitated by phosphorylation but it is not known if only the first loop, only the third loop, or both the first and third loops need to be phosphorylated for this response.

Amino Acid Sequence↗

PubMed1997

beta2-adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists.

Previous studies indicated that partial agonists cause less desensitization of the beta2-adrenergic receptor (betaAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of betaAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the betaAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified betaAR behaved identically to the wild type receptor with regard to agonist Kd, coupling efficiency, and desensitization. The coupling efficiencies for betaAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0-30 min) of agonist-induced desensitization of betaAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine >/= fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the betaAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, betaAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of betaAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of betaAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 +/- 0.82) was not appreciably different from ephedrine (5.0 +/- 0.96) and significantly less than that caused by albuterol (10.4 +/- 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of betaARK phosphorylation of the betaAR.

Adenylyl Cyclases↗

PubMed1997

Agonist-induced desensitization, internalization, and phosphorylation of the sst2A somatostatin receptor.

Cellular responsiveness to the inhibitory peptide somatostatin (SRIF) or its clinically used analogs can desensitize with agonist exposure. While desensitization of other seven-transmembrane domain receptors is mediated by receptor phosphorylation and/or internalization, the mechanisms mediating SRIF receptor (sst) desensitization are unknown. Therefore, we investigated the susceptibility of the sst2A receptor isotype to ligand-induced desensitization, internalization, and phosphorylation in GH-R2 cells, a clone of pituitary tumor cells overexpressing this receptor. A 30-min exposure of cells to either SRIF or the analog SMS 201-995 (SMS) reduced both the potency and efficacy of agonist inhibition of adenylyl cyclase. Internalization of receptor-bound ligand was rapid (t1/2 = 4 min) and temperature-dependent. SRIF and SMS increased the phosphorylation of the 71-kDa sst2A protein 25-fold within 15 min. Receptor phosphorylation was dependent on both the concentration and time of agonist exposure and was not affected by pertussis toxin pretreatment, indicating that receptor occupancy rather than second messenger formation was required. Receptor phosphorylation was also stimulated by phorbol 12-myristate 13-acetate activation of protein kinase C. Both ligand-stimulated and phorbol 12-myristate 13-acetate-stimulated receptor phosphorylation occurred primarily on serine. These studies are the first demonstration of agonist-dependent desensitization, internalization, and phosphorylation of the sst2A receptor and suggest that phosphorylation may mediate the homologous and heterologous regulation of this receptor.

Adenylate Cyclase Toxin↗

PubMed1997

Immunohistochemical localization of somatostatin receptor SST2A in the rat pancreas.

Somatostatin (SRIF) acts on specific membrane receptors to inhibit exocrine and endocrine pancreatic functions. Five SRIF receptor genes have been cloned, producing six receptor proteins (sst-s). We used a recently developed antibody to localize the sst2A splice variant in the rat pancreas. Western blots identified the sst2A receptor as an 90 kDa glycosylated protein in pancreatic tissue. In tyramide-amplified immunostainings all acinar cells, and the glucagon and pancreatic polypeptide immunoreactive cells (A and PP, respectively) were intensely labeled for sst2A, while no signal was detected in SRIF producing (D) cells. A very few insulin immunoreactive (B) cells were also labeled for sst2A, but the signal in these cells was lower than in exocrine, A or PP cells. Absorption of the sst2A antibody with the receptor peptide abolished specific staining in both immunoblots and tissue sections (negative control). These studies are the first to localize any SRIF receptor subtype in the rat pancreas. The specific localization of sst2A receptor in acinar, A and PP cells if confirmed in humans, would suggest that subtype specific analogs will be useful for the therapeutic regulation of exocrine and/or endocrine pancreatic secretion.

Animals↗

PubMed1997

Immunohistochemical localization of somatostatin receptor SST2A in the rat pancreas.

Alternative Splicing↗

PubMed1996

Progressive cytoplasmic tail truncations of the lutropin-choriogonadotropin receptor prevent agonist- or phorbol ester-induced phosphorylation, impair agonist- or phorbol ester-induced desensitization, and enhance agonist-induced receptor down-regulation.

Stably transfected human kidney 293 cells expressing the wild type rat LH/CG receptor (rLHR) or receptors with C-terminal tails truncated at residues 653, 631, or 628 (designated rLHR-t653, rLHR-t631, and rLHR-t628) were used to probe the importance of this region on the regulation of hormonal responsiveness. The chosen cells line express comparable densities of cell surface rLHR, bind human CG (hCG) with high affinity, and respond to hCG with increases in cAMP and inositol phosphate accumulation. Cells expressing rLHR-wt or rLHR-t653 responded to hCG, or phorbol 12-myristate-13-acetate (PMA) stimulation with a similar increase in rLHR phosphorylation. Neither of these two stimuli increased rLHR phosphorylation in cells expressing rLHR-t631 or rLHR-t628, however. The cell line expressing rLHR-t653, the phosphorylation-positive receptor mutant, desensitized normally in response to PMA or hCG stimulation. This truncated form of rLHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing rLHR-t631 or rLHR-t628, the two phosphorylation-negative receptor mutants, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the rLHR are involved in PMA-induced desensitization, hCG-induced de-sensitization, and hCG-induced down-regulation. These results also establish a positive correlation between rLHR phosphorylation and desensitization and a negative correlation between rLHR phosphorylation and down-regulation.

Binding Sites↗

PubMed1995

Truncation of the C-terminal tail of the follitropin receptor does not impair the agonist- or phorbol ester-induced receptor phosphorylation and uncoupling.

We have recently shown that addition of follitropin (FSH) or a phorbol ester (phorbol 12-myristate 13-acetate (PMA)) to cells expressing the recombinant follitropin receptor (FSHR) results in both phosphorylation and uncoupling of the FSHR from adenylyl cyclase. In the light of findings reported with other G protein-coupled receptors we have proposed that phosphorylation of the FSHR mediates the uncoupling from adenylyl cyclase. The experiments described herein represent the first attempt to determine the location of the amino acid residues that become phosphorylated in FSHR and to test the hypothesis that phosphorylation is responsible for uncoupling of FSHR from adenylyl cyclase. As a first step in identifying which residues may be phosphorylated in response to hFSH and PMA, we constructed a mutant of the FSHR cDNA in which the C-terminal cytoplasmic tail was truncated at residue 635 (FSHR-t635), thus removing all but one of the potential phosphorylation sites present in the C-terminal tail. Cells expressing FSHR-t635 bind hFSH with the appropriate affinity and respond with increases in cAMP and inositol phosphate accumulation. The maximal cAMP and inositol phosphate responses of cells expressing FSHR-t635 are higher than those of cells expressing the wild type FSHR, but the concentration of hFSH required to elecit these responses is similar in both cell lines. Immunoprecipitation of FSHR-t635 shows that the truncated receptor is still effectively phosphorylated in response to hFSH or PMA. Phosphoamino acid analysis reveals that, like the wild-type FSHR, FSHR-t635 phosphorylation occurs on serine and threonine residues. Peptide mapping suggests that the phosphorylated residues in the FSHR and FSHR-t635 are located within the same areas of the intracellular regions of the receptors. In addition to stimulating phosphorylation of FSHR-t635, hFSH and PMA also effectively uncouple the truncated receptor from adenylyl cyclase. Taken together, these data show that hFSH and PMA can both phosphorylate and uncouple a FSH receptor species with a cytoplasmic tail truncated at residue 635.

Adenylyl Cyclases↗

PubMed1995

Human chorionic gonadotropin (CG)- and phorbol ester-stimulated phosphorylation of the luteinizing hormone/CG receptor maps to serines 635, 639, 649, and 652 in the C-terminal cytoplasmic tail.

In a number of instances, binding of a ligand to its receptor results in receptor phosphorylation that mediates receptor uncoupling from its effector. Recently, we showed that human CG (hCG)- or phorbol ester- [phorbol 12-myristate-13-acetate (PMA)] stimulation of cells transfected with the LH/CG receptor induced rapid LH/CG receptor phosphorylation and a reduced cAMP response upon reexposure to hCG. The fact that hCG and PMA both phosphorylate and uncouple the LH/CG receptor suggests a common mechanism of action, namely the activation of protein kinase C. The studies presented here were designed to investigate the role of the C kinase in LH/CG receptor phosphorylation and to locate the phosphorylation site(s) within the receptor protein. The experiments presented here show that although hCG activates the C kinase in these cells, phosphorylation of the LH/CG receptor in response to hCG is maintained in C kinase-deficient cells. This suggests that activation of protein kinase C is not required for hCG-induced phosphorylation of its receptor. As a first step in locating the phosphorylation sites within the receptor polypeptide, we performed phosphoamino acid analysis of the phosphorylated LH/CG receptor. Only phosphoserine residues were detected. Based on the assumption that the phosphoserine(s) must be located within the intracellular regions of the receptor, we isolated cell lines expressing the wild type LH/CG receptor or receptors with cytoplasmic tails truncated at residue 653 or 631.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Follitropin (FSH) and a phorbol ester stimulate the phosphorylation of the FSH receptor in intact cells.

Using a cell line stably transfected with the rat follitropin (FSH) receptor cDNA we demonstrate that the FSH receptor becomes phosphorylated when cells are exposed to FSH. Since binding of FSH to its receptor results in an increase in cAMP and inositol phosphate accumulation, we examined the potential involvement of protein kinase A and C in mediating receptor phosphorylation. Stimulation of protein kinase A does not appear to be necessary because hFSH-induced receptor phosphorylation was minimally impaired in a cell line that overexpresses cAMP phosphodiesterase. Moreover, stimulation of the protein kinase A pathway with other agonists result in minimal phosphorylation of the FSH receptor. Stimulation of the protein kinase C with a phorbol ester did result in an increase in receptor phosphorylation, and down-regulation of the protein kinase C decreased, but did not abolish, the FSH-induced receptor phosphorylation. The possible impact of phosphorylation on the functions of the receptor was examined by testing if conditions that lead to phosphorylation decrease the ability of FSH to stimulate cAMP synthesis. Our data show that as with the addition of FSH, addition of a phorbol ester also results in a decrease in the ability of FSH to stimulate cAMP synthesis.

Adenylyl Cyclases↗