Receptor Replenishment Research Articles

Cannabinoid receptor interacting protein suppresses agonist-driven CB1 receptor internalization and regulates receptor replenishment in an agonist-biased manner.

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1 R) distal C-terminus-associated protein that modulates CB1 R signaling via G proteins, and CB1 R down-regulation but not desensitization (Blume etal. [2015] Cell Signal., 27, 716-726; Smith etal. [2015] Mol. Pharmacol., 87, 747-765). In this study, we determined the involvement of CRIP1a in CB1 R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB1 Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1 R and CRIP1a, and exhibits a well-characterized endocannabinoid signaling system. We developed stable CRIP1a-over-expressing and CRIP1a-siRNA-silenced knockdown clones to investigate gene dose effects of CRIP1a on CB1 R plasma membrane expression. Results indicate that CP55940 or WIN55212-2 (10nM, 5 min) reduced cell surface CB1 R by a dynamin- and clathrin-dependent process, and this was attenuated by CRIP1a over-expression. CP55940-mediated cell surface CB1 R loss was followed by a cycloheximide-sensitive recovery of surface receptors (30-120min), suggesting the requirement for new protein synthesis. In contrast, WIN55212-2-mediated cell surface CB1 Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10nM)-mediated increase in cell surface CB1 Rs, which is postulated to be as a result of rimonabant effects on 'non-agonist-driven' internalization. These studies demonstrate a novel role for CRIP1a in agonist-driven CB1 R cell surface regulation postulated to occur by two mechanisms: 1) attenuating internalization that is agonist-mediated, but not that in the absence of exogenous agonists, and 2) biased agonist-dependent trafficking of de novo synthesized receptor to the cell surface.

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.

Covering a Broad Dynamic Range: Information Processing at the Erythropoietin Receptor

Cell surface receptors convert extracellular cues into receptor activation, thereby triggering intracellular signaling networks and controlling cellular decisions. A major unresolved issue is the identification of receptor properties that critically determine processing of ligand-encoded information. We show by mathematical modeling of quantitative data and experimental validation that rapid ligand depletion and replenishment of the cell surface receptor are characteristic features of the erythropoietin (Epo) receptor (EpoR). The amount of Epo-EpoR complexes and EpoR activation integrated over time corresponds linearly to ligand input; this process is carried out over a broad range of ligand concentrations. This relation depends solely on EpoR turnover independent of ligand binding, which suggests an essential role of large intracellular receptor pools. These receptor properties enable the system to cope with basal and acute demand in the hematopoietic system.

Role of the rate of internalization of the agonist-receptor complex on the agonist-induced down-regulation of the lutropin/choriogonadotropin receptor.

The extent of agonist-induced down-regulation of the LH/CG receptor (LHR) in human kidney 293 cells transfected with the rat LHR (rLHR) is much lower than in two Leydig tumor cell lines (MA-10 and R2C) that express the rodent LHR endogenously. This difference can not be attributed to differences in the recycling of internalized receptors, or in the replenishment of new receptors at the cell surface. It can be correlated, however, with the half-life of internalization of the bound agonist, which is approximately 60 min in Leydig tumor cells and about 100 min in transfected 293 cells. To determine whether the rate of internalization of the bound agonist affects down-regulation, we compared these two parameters in 293 cells expressing four rLHR mutants that enhance internalization and three mutants that impair internalization. We show that all four mutations of the rLHR that enhanced internalization enhanced down-regulation, while only one of the three mutations that impaired internalization impaired down-regulation. In addition, cotransfections of 293 cells with the rLHR-wt and three constructs that enhanced internalization (G protein-coupled receptor kinase 2, beta-arrestin, and arrestin-3) increased down-regulation, while a related construct (visual arrestin) that had no effect on internalization also had no effect on down-regulation. We conclude that the rate of internalization of the agonist-LHR complex is the main determinant of the extent of down-regulation of the LHR.

Leukocyte polarization in cell migration and immune interactions.

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.

Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor.

The receptor for the protease thrombin is a member of the G protein-coupled receptor family, but is activated by a unique proteolytic mechanism. The irreversibility of this proteolytic mechanism and the fact that the ligand is tethered to its receptor raise special questions about inactivation of cleaved receptors and recovery of thrombin responsiveness. We compared the intracellular trafficking of the thrombin receptor to that of the beta 2-adrenergic receptor in transfected Rat1 fibroblasts. In unstimulated cells almost all beta 2 receptors were located on the plasma membrane; by contrast, part of a cell's thrombin receptors were found in an intracellular membrane compartment which co-localized with Golgi markers. Stimulation by agonist caused internalization and subsequent recycling of the beta 2-adrenergic receptor, but most activated thrombin receptors were internalized and targeted to lysosomes. The intracellular pool of thrombin receptors found in unstimulated cells was protected from activation by thrombin, but was translocated to the plasma membrane upon activation of cell surface thrombin receptors. Replenishment of plasma membrane thrombin receptors correlated with recovery of thrombin responsiveness. These observations reveal a novel trafficking mechanism for resensitizing the thrombin receptor as opposed to the internalization/recycling pathway of other G protein-coupled receptors.

Role of microsomal receptors in steroid hormone action

Ventral prostate was used as a system to study the nature and properties of microsomal androgen receptor. The endoplasmic reticulum from rat ventral prostate contains high-affinity, low-capacity binding sites for androgen that are intrinsic to this intracellular compartment. Microsomal androgen receptors are not due to plasma membrane or cytosol contamination and they display a fast turnover, with depletion after 1 hour and complete replenishment 6 hours after androgen stimuli. Cycloheximide, but not actinomycin D, inhibits microsomal androgen receptor replenishment, indicating that testosterone may control microsomal receptor levels acutely by posttranscriptional mechanisms. Microsomal androgen receptor is a 5S protein that has a higher stability than its cytosolic counterpart, regardless of the presence of ligand. It does not become activated after heat or salt treatment. After extraction of binding sites, microsomes are capable of accepting cytosol mibolerone-receptor complexes to a level similar to the concentration of depleted binding sites; microsomes from nontarget tissues do not manifest such capability. The results indicate the coexistence of a non-DNA-binding form of androgen receptor in the microsomal membranes with the typical DNA-binding form of androgen receptor present in the cytosol of ventral prostate homogenates. Microsomal androgen receptor may represent an additional level of regulation of androgen action in the intact target cell.

Cellular mechanisms of the clearance function of type C receptors of atrial natriuretic factor.

Clearance (C) receptors of atrial natriuretic factor (ANF) have an important role of removing ANF from the circulation. In the present study we investigated the cellular mechanisms of this function. 125I-ANF1-28 specifically bound to C-ANF receptors in cultured bovine vascular smooth muscle cells is internalized by a temperature-dependent process at a rate of 5% occupied receptors/min at 37 degrees C. Internalized 125I-ANF1-28 is rapidly metabolized and released to the medium as [125I]monoiodotyrosine, a process that is reversibly inhibited by NH4Cl (10 mM). Retroendocytosis of receptor-ligand complex is detectable when intracellular accumulation of the ligand is maximized by previous incubation with NH4Cl. In the presence of saturating concentrations of 125I-ANF1-28 when cells are warmed from 4 to 37 degrees C, there is first a decrease and then an almost complete replenishment of surface C-ANF receptors, a phenomenon that is not altered by protein synthesis inhibition with cycloheximide. In the absence of temperature transition, the density of surface C-ANF receptors remains constant, and by 2 h of incubation the amount of ANF hydrolyzed far exceeds the total amount of C-ANF receptors. The total population of surface C-ANF receptors is internalized and recycled every hour, and these processes are constitutive since they also occur in the absence of ligand. Trypsinization and solubilization experiments further indicate that C-ANF receptors are rapidly internalized and recycled to the cell membrane. The results demonstrate that receptor-ligand internalization, extensive receptor recycling, and lysosomal hydrolysis of ANF are the cellular mechanisms by which C-ANF receptors exert their clearance function.

Estradiol and chlordecone interactions with the estradiol receptor

The in vivo and in vitro effects of the chlorinated pesticide, chlordecone, on the estradiol receptor of adult ovariectomized CDF-344 rats were examined. Chlordecone's competition with [ 3H]estradiol for binding to the estradiol receptor in vitro was similar whether receptors were derived from neural or uterine tissue. In vivo, chlordecone led to nuclear retention of estradiol receptors in both tissues, but in comparison to estradiol, the latency for such retention was slower following chlordecone. Nevertheless, chlordecone- and estradiol-treated animals eventually achieved the same degree of nuclear retention in uterine tissue. In brain, estradiol-like levels of nuclear retention were never found in chlordecone-treated females. Estradiol receptor replenishment following chlordecone or estradiol was similar in uterine tissue but not in brain. Chlordecone's lesser ability to mimic estradiol's receptor dynamics in the brain may contribute to the apparent tissue differences in chlordecone's estrogenicity.

Rapid Leydig cell proliferation and luteinizing hormone receptor replenishment in the neonatal rat testis after a single injection of human chorionic gonadotropin.

Two-day-old rats were stimulated with a single dose of human chorionic gonadotropin (hCG). Changes in the Leydig cell number, mitotic activity, cell size, and number of luteinizing hormone (LH) receptors were studied. The Leydig cell number of the hCG-treated animals was 1.8 times that of the control on Day 1 and remained elevated for the rest of the 5-day experiment (p less than 0.0001). On Day 1 the number of Leydig cell mitoses in the hCG group was greater (p less than 0.05) than in the controls. The Leydig cell size increased transiently to two times that of the control (p less than 0.01) within the first day after the treatment and returned to control size by Day 5. The number of LH receptors per testis decreased 81% in 1 day (p less than 0.01), but returned to control level by Day 3. Since Leydig cell numbers were constant after Day 1, the rapid receptor recovery was obviously due to restoration of the binding sites rather than increased cell number. The present results demonstrate a rapid proliferative response and rapid LH receptor replenishment in the fetal-neonatal Leydig cells after gonadotropic stimulation. These responses of fetal-type Leydig cells are in clear contrast to those observed in adult testes after a similar stimulation.

Slow regeneration of inmune complex binding sites on the macrophage surface after previous digestion with pronase

Peroxidase-antiperoxidase (PAP) binding sites on the rat peritoneal macrophage surface were damaged by pronase digestion and the reappearance of functionally intact receptors was investigated morphologically and spectrophotometrically. Pronase digestion decreased the PAP binding ability of macrophages to about 40% of the original value. Removing the pronase the regeneration of ligand binding was very protracted with only about 60% intact receptors even after 30 min. From these findings we conclude that the recycling of internalized Fc receptors greatly contribute to the replenishment of receptors on the cell surface.

Mechanism of replenishment of androgen receptors in cytosol of mouse submandibular gland.

Female mice were used to examine the process of depletion and replenishment of cytosolic androgen receptors in submandibular glands, and to investigate the effects of cycloheximide and actinomycin D on these processes. The dose-dependence of receptor depletion and replenishment in the cytosolic fraction, and that of receptor accumulation in the nuclear fraction were investigated. Almost 100% depletion was revealed 1 h after the injection of testosterone propionate at a dose of 500 or 50 micrograms testosterone/100 g body weight. With a 5 micrograms dose, depletion of cytosolic receptors was not complete and replenishment proceeded rapidly compared with that which occurred with the 50 or 500 micrograms dose. The nuclear receptor level increased 1 h after injection of testosterone, and the raised level was gradually reduced to the pretreatment level with all doses. However, the time required for this return to pretreatment level was dependent on the dose of testosterone. The change in the levels of cytosolic and nuclear androgen receptors following injection of testosterone was parallel to the level of circulating androgen. To determine the requirements for transcriptional and translational events in the replenishment process, cycloheximide and actinomycin D were given in vivo. The process of replenishment of cytosolic receptors was inhibited by the injection of cycloheximide. However, actinomycin D exerted no inhibitory effect on receptor replenishment. Neither cycloheximide nor actinomycin D had any effect on the nuclear receptor level until 6 h after the injection of testosterone. Cycloheximide or actinomycin D alone had no effect on the cytosolic or nuclear receptor level. These results suggest that receptor replenishment involves protein synthesis.

Rapid recovery of gonadotroph function after down-regulation of receptors for GnRH in ewes.

Several characteristics of the hypothalamo-hypophysial axis were examined after down-regulation of GnRH receptors and the desensitization which accompanies it in the ewe. Down-regulation of GnRH receptors, induced by i.v. infusion of GnRH (2.5 micrograms/h) for 24 h, resulted in a 50% decrease in the number of receptors for GnRH at the end of the infusion period. The number of receptors for GnRH was restored to control values by 6 h after the infusion ended and remained stable at 12, 24, 48, 72 and 96 h after infusion. The amount of LH released in response to an i.v. injection of 100 micrograms GnRH was reduced by 82% at the end of the infusion period, but there was no significant reduction in the GnRH-induced release of FSH. The GnRH-induced release of LH was restored by 12 h after the infusion ended; however, the amount of FSH released in response to GnRH was not different from control values at any time. A decrease in both the amplitude and frequency of endogenous pulses of LH was observed from 0 to 12 h after the end of the infusion period. At no time did the concentration of gonadotrophins in the pituitary change. These results demonstrate that replenishment of receptors for GnRH and recovery of the ability of the gonadotroph to release LH are associated events. However, the GnRH-induced release of FSH does not appear to be closely related to the number of GnRH receptors. We suggest that continuous exposure to GnRH may inhibit the hypothalamic pulse generator as well as the pituitary response to the pulse generator.

1,25-Dihydroxyvitamin D 3 receptor replenishment in the chicken intestine

1,25-Dihydroxyvitamin D 3 intestinal receptor replenishment was examined in rachitic chickens after hormone administration. A single injection of 1,25-dihydroxyvitamin D 3 caused an increase in the level of occupied receptors with a concomitant decrease in the amount of unoccupied receptors. Maximum occupancy occurred 1 h after hormone injection. The metabolic inhibitor of protein synthesis, cycloheximide, was employed to obtain additional information concerning the fate of 1,25-dihydroxy vitamin D 3 receptor complexes. Cycloheximide, at a dose that effectively blocked protein synthesis, had no effect on the time-course or the magnitude of replenishment of nuclear receptors. Additionally, repletion with vitamin D 3 or administration of several injections of 1,25-dihydroxy vitamin D 3 did not lead to a lag in replenishment time or a significant decrease in total receptor levels. These findings demonstrate that recycling of receptors plays an important functional role for the replenishment of unoccupied 1,25-dihydroxyvitamin D 3 intestinal receptors.

Progesterone receptor replenishment during sustained progesterone treatment in the hamster uterus.

Previous studies have demonstrated that uterine progesterone (P) receptor is under dual hormonal control; estradiol (E2) induces the synthesis of cytosolic P receptor, and P induces the loss of its own receptor and antagonizes E2-induced P receptor replenishment. The objective of this study was to examine the contributions of E2 and P in the replenishment of uterine P receptor during sustained P exposure. Silastic implants of varying length (0.4, 0.8, 1.5, 2.5, and 5.0 cm) were packed with crystalline P and placed sc in the flank region of ovariectomized adult female hamsters. The serum P levels obtained with these implants (2-22 ng/ml) were within the physiological range observed previously during the estrous cycle, pregnancy, and lactation in the Syrian hamster. Control animals received a blank implant (1.5 cm). Three, 5, and 7 days after placement of the implants, uterine cytosolic and nuclear P receptor levels were decreased as serum P level was increased by the P implants. Total cellular P receptor level was inversely correlated with serum P level at 3 days (r = -0.996), 5 days (r = -0.98), and 7 days (r = -0.99). To distinguish the effect of E2 and P, ovariectomized animals were maintained on Silastic implants of P (1.5 cm) or P plus E2 (1.0 cm). After 3 days, cytosolic P receptor was determined 0, 8, 16, 24, and 48 h after removal of P implants. No difference was observed in cytosolic P receptor between P and P plus E2 groups before P withdrawal. E2-maintained animals showed a significant rise of cytosolic P receptor at all time points after P withdrawal. Although P withdrawal in the absence of E2 showed no significant change in P receptor at 8 or 16 h, a significant increase in P receptor (equivalent to that of ovariectomized control animals) was observed at 24 and 48 h. Treatment of ovariectomized animals with cycloheximide significantly reduced uterine cytosolic P receptor levels 8, 18, and 24 h after treatment. These results suggest that 1) an active receptor replenishment process occurs in the absence of E2; 2) this replenishment process does not appear to be P dependent, but, rather, constitutively expressed; and 3) the rate of constitutive P receptor replenishment is slower and of lower magnitude than that promoted by E2. Because P antagonizes E2-induced P receptor, a constitutive P receptor replenishment mechanism may play an important role in the maintenance of P action during sustained P exposure, such as in pregnancy.

Depletion-replenishment of the testicular estrogen receptor: Sensitivity to cycloheximide and actinomycin D

Male rats (30–35 days old) were utilized to examine the process of depletion and replenishment of the testicular cytosolic estrogen receptor and to investigate the effects of cycloheximide and actinomycin D on these processes. The dose dependence and temporal nature of receptor depletion and replenishment were investigated. Maximum depletion (> 90%) occurred by 1 h after in vivo administration of either 5 or 10 μg estradiol-17β. Depletion of the cytosolic receptor at 1 h occurred concomitant to a marked increase in nuclear receptor thus indicating translocation. Receptor replenishment to control levels was observed by 6 h post treatment. To determine the requirements for transcriptional and translational events in the replenishment process, actinomycin D and cycloheximide were administered in vivo. Simultaneous treatment with cycloheximide and estradiol resulted in a significant inhibition of replenishment at 6 and 12 h post treatment of 46 and 60% below control levels, respectively. Cycloheximide treatment alone had no effect on receptor levels. A significant inhibition of replenishment at 6 h was also shown when cycloheximide was given 3 h after estradiol treatment. Cycloheximide administration at 6 h after estradiol significantly suppressed receptor levels at 12 h suggesting that replenished receptor levels at 6–12 h are in a state of rapid turnover. Receptor replenishment exhibited a different response to actinomycin D treatment in that significant inhibition was observed only when the drug was administered at 3 h after estradiol treatment. These results demonstrate that testicular cytosolic estrogen receptor depletion is dose and time dependent. The results further demonstrate that receptor replenishment involves protein synthesis and suggests that synthesis of new RNA might also be required.

Apparently normal oestrogen receptor system in ovariectomized ewes with impaired response to oestrogen after prolonged grazing on oestrogenic pasture

Functional abnormalities have been observed previously in the oestrogen-responsive tissues of ovariectomized ewes with permanent infertility resulting from prolonged exposure to oestrogenic pastures. In the present study, such ewes had higher rates of protein and glycoprotein synthesis in the cervix and uterus than control ewes, but the number of oestradiol-17 beta receptors in the nucleus was similar in each group. After treatment with oestradiol-17 beta, the increase in synthesis of protein and glycoprotein in the uterus and cervix was less in clover-affected ewes, but the amount of oestradiol-17 beta-receptor complex in the nucleus of uterine cells 6 h after ewes were injected with oestradiol-17 beta was similar in clover-affected and control ewes. The rate of replenishment of oestradiol-17 beta receptors in the cytoplasm at 24 and 48 h after oestradiol-17 beta injection was also similar in both groups. The abnormal function seen in the genital tract of clover-affected ewes could not, therefore, be shown to depend on changes in oestradiol-17 beta receptors.

Temporal and receptor correlates of the estrogen response in sheep

Uterine blood flow and uterine cytosol and nuclear estrogen receptors were measured at critical times during estradiol-induced vasodilatation in acute anesthetized and chronic conscious sheep preparations at estradiol bolus injection frequencies from 1 to 24 hours. During acute experiments, the uterine blood flow response was muted and cytosol estrogen receptor replenishment did not occur whereas full replenishment occurred in 12 hours in conscious ewes. In ewes treated with daily estradiol that had a stable daily uterine blood flow response, the uterine blood flow response 24 hours after uterine biopsy was similar to the preoperative one. Analysis of the duration of peak uterine blood flow levels and the rate of uterine blood flow descent from peak levels showed that an interval of 18 hours between estradiol injections was necessary for the uterine blood flow response to approximate that observed after 24 hours. These observations suggest: (1) that uterine vascular receptor replenishment is delayed compared with that of the total uterus; (2) that operative stress compromises cytosol estrogen receptor metabolism and possibly nuclear estrogen receptor function; (3) that the delayed maximum uterine blood flow response to estradiol in ewes previously untreated with estradiol is due to a trophic uterine effect of daily estradiol stimulation.

Progestin-induced down regulation of nuclear estrogen receptor in uterine decidual cells: Analysis of receptor synthesis and turnover by the density-shift method

The density-shift method was used to study the effect of the synthetic progestin, R5020, (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) on the turnover and synthesis of nuclear estrogen receptor in hamster decidual cells. Newly-synthesized receptor was labeled with dense [ 2H, 13C, 15N] amino acids and separated from pre-existing receptor by density-gradient centrifugation. Progestin increased receptor turnover within 3 h of treatment and blocked estradiol-induced receptor synthesis at 6 h and 9 h. Thus, progestin down regulates estrogen receptor by increasing receptor turnover and inhibiting estrogen-induced receptor replenishment.

Relationship of estrogen receptors and protein synthesis to the mitogenic effect of estrogens.

1,3,5(10)-Estratriene-3,16 alpha-diol (16 alpha-E2) is an estrogen which is ineffective in stimulating DNA synthesis in the prepuberal rat uterus when administered to rats in a single injection in doses up to at least 5 micrograms. However, it acquires the same high mitogenic potency as 1,3,5(10)-estratriene-3,17 beta-diol (17 beta-E2) if a 5-micrograms dose is administered over a 12-h period via sequential injections of 1 microgram each at 3-h intervals. In an attempt to explain this phenomenon we have found that the ability of an estrogen to maintain a stimulated rate of protein synthesis for 12 h correlates directly with its ability to stimulate DNA synthesis. A single injection of 16 alpha-E2 stimulates protein synthesis at 4 h to a degree comparable to 17 beta-E2. By 12 h when the effect of 17 beta-E2 is maximal, the effect of one injection of 16 alpha-E2 has diminished to control levels. However, if 16 alpha-E2 is administered via sequential injections at 3-h intervals, protein synthesis at 12 h is stimulated to the same extent achieved by a single injection of 17 beta-E2. We also have examined the fate of estrogen receptors in relationship to these changes in protein and DNA synthesis. The effects of a single injection of 16 alpha-E2 differ in four respects from those of 17 beta-E2: 1) the retention of receptors in the nuclear form is shorter; 2) replenishment of receptors to the cytosolic form is more rapid and greater than 80% complete within 3 h; 3) fewer receptors are processed, i.e. the loss of receptors detectable by exchange assay is smaller; and 4) the overshoot in receptor replenishment 24 h after an injection is smaller. Overall, the stimulation of DNA synthesis is positively related to the rate of protein synthesis 12 h after an injection of estrogen, the retention of receptors in the nuclear form, and the amount of receptor processing.