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P W Fletcher

Publications and source records attributed to P W Fletcher.

At least 19 recordsLinked to original sources
PubMed2001

A surrogate-based approach for post-genomic partner identification.

BACKGROUND: Modern drug discovery is concerned with identification and validation of novel protein targets from among the 30,000 genes or more postulated to be present in the human genome. While protein-protein interactions may be central to many disease indications, it has been difficult to identify new chemical entities capable of regulating these interactions as either agonists or antagonists. RESULTS: In this paper, we show that peptide complements (or surrogates) derived from highly diverse random phage display libraries can be used for the identification of the expected natural biological partners for protein and non-protein targets. Our examples include surrogates isolated against both an extracellular secreted protein (TNFbeta) and intracellular disease related mRNAs. In each case, surrogates binding to these targets were obtained and found to contain partner information embedded in their amino acid sequences. Furthermore, this information was able to identify the correct biological partners from large human genome databases by rapid and integrated computer based searches. CONCLUSIONS: Modified versions of these surrogates should provide agents capable of modifying the activity of these targets and enable one to study their involvement in specific biological processes as a means of target validation for downstream drug discovery.

Computational Biology↗
PubMed1999

The recombinant third domain of human alpha-fetoprotein retains the antiestrotrophic activity found in the full-length molecule.

Previous studies have shown that alpha-fetoprotein (AFP) interferes with estrogen (E2)-stimulated growth, including E2-stimulated breast cancer growth. In an effort to localize the antiestrotrophic portion of the molecule, the C-terminal one-third (200 amino acids) of human AFP, known as Domain III, was produced in a baculovirus expression system as a fusion protein containing an amino terminal histidine tag. The histidine tag was included to facilitate purification by metal ion affinity chromatography. The purified recombinant Domain III fusion protein was functionally similar to full-length natural AFP isolated from human cord sera or from cultured human hepatoma cells (HepG2) in that they all produced significant and quantitatively similar inhibition of E2-stimulated growth of immature mouse uterus. Furthermore, the dose-response profiles of the recombinant Domain III AFP and natural full-length AFP were similar. Preincubation of either protein in a molar excess of E2 lowered the minimally effective antiestrotrophic dose and produced a difference spectrum consistent with a change in conformation. These findings indicate that the antiestrotrophic activity of AFP is contained within the third domain of the molecule, and they have obvious implications for the production of biologically active peptides derived from this portion of the AFP molecule.

Animals↗
PubMed1997

Bradykinin- and thrombin-induced increases in endothelial permeability occur independently of phospholipase C but require protein kinase C activation.

We determined whether activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and a subsequent increase in cytosolic calcium concentration ([Ca2+]i) was an obligatory signaling event mediating the increase in transendothelial permeability induced by bradykinin (BK) and alpha-thrombin (alpha-T). Both BK and alpha-T (each at a concentration range of 0.01-1 microM) caused dose-dependent increases in transendothelial 125I-albumin permeability in cultured bovine pulmonary artery endothelial cell monolayers. Both agonists also produced a rise in inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] by 10 sec that was followed by a prolonged increase in [Ca2+]i. Pretreatment of endothelial cells with the PLC inhibitor, 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dion [(U73122) at 10 microM for 15 min], prevented the increases in Ins(1,4,5)P3 and [Ca2+]i induced by both BK and alpha-T. However, inhibition of PLC with U73122 or another PLC inhibitor, neomycin, did not prevent the increase in endothelial permeability induced by either agonist. In contrast, depletion of cellular protein kinase C (PKC) with phorbol-12-myristate 13-acetate (0.01 microM for 20 hr) increased both BK- and alpha-T-induced phosphoinositide turnover but inhibited the agonist-induced increase in permeability. A PKC inhibitor, staurosporine (5 microM) likewise inhibited the BK-induced increase in endothelial cell permeability to albumin. We conclude that increases in endothelial permeability induced by the inflammatory mediators, BK and thrombin, can occur independently of PLC activation and increased [Ca2+]i but that a PKC-dependent pathway is required for the permeability response.

Animals↗
PubMed1993

Follicle-stimulating hormone increases the turnover of G-protein alpha i-1- and alpha i-2-subunit messenger RNA in Sertoli cells by a mechanism that is independent of protein synthesis.

We have shown previously that FSH differentially regulated G-protein alpha i-subunit mRNA levels in primary cultures of rat Sertoli cells. The mechanism by which FSH regulated the steady state levels of these mRNAs was investigated by assessing the effect of FSH on G-protein alpha-subunit mRNA stability and the potential involvement of newly synthesized proteins. The half-life (t 1/2) of alpha-subunit mRNA was determined by inhibiting Sertoli cell transcription with actinomycin-D and quantifying alpha-subunit mRNA levels by Northern blot analyses. Transcripts for alpha i-2 and alpha 3 were extremely stable, possessing t1/2 of 44 and 51 h, respectively. In contrast, the t1/2 of alpha i-3 mRNA was only 3.6 h. Turnover of alpha i-1 mRNA occurred as a two-phase decay, with an initial t1/2, fast of 0.8 h, followed by a second phase with t1/2, slow of 11.1 h. Treatment of Sertoli cells with FSH in the presence of actinomycin-D destabilized the alpha i-1 mRNA to a single phase decay with a t1/2 of 5.7 h. FSH also decreased the stability of the alpha i-2 mRNA from a t1/2 of 51 h to a t1/2, fast of 16.5 h. These effects of FSH on alpha i-1 and alpha i-2 mRNA stability may contribute to the ability of FSH to decrease steady state alpha i-1 and alpha i-2 mRNA levels. The effect of FSH on alpha i-3 mRNA stability was not significant, suggesting an alternative mechanism regulating the FSH-mediated increase of alpha i-3 mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Membrane-bound and solubilized brain 5HT3 receptors: improved radioligand binding assays using bovine area postrema or rat cortex and the radioligands 3H-GR65630, 3H-BRL43694, and 3H-LY278584.

Bovine area postrema tissue was used as a convenient source of tissue for studies of brain 5HT3 receptors. 5HT3 receptor density was determined to be 97 +/- 5 fmol/mg and 124 +/- 10 fmol/mg of protein using the commercially available 5HT3 radioligands, 3H-GR65630 and 3H-BRL43694, respectively. The equilibrium dissociation constants (KD) for 3H-GR65630 and 3H-BRL43694 were 0.5 +/- 0.1 nM and 1.7 +/- 0.3 nM, respectively. The affinities of a series of drugs for the 5HT3 receptor using the two radioligands were essentially identical; the Ki values and order of affinities of agonists and antagonists were very similar to data published in studies on radiolabeling of 5HT3 receptors in rat brain. 3H-LY278584 also labeled 5HT3 receptors in bovine area postrema homogenates with a KD of 3.1 +/- 0.1 nM and a Bmax of 84 +/- 6 fmol/mg. In rat cortical homogenates, 3H-LY278584 produced the most reliable specific signal of 72%, with a KD of 2.6 +/- 0.3 nM and a Bmax value of 10.5 +/- 1 fmol/mg. At 1 nM, 3H-GR65630 or 3H-BRL43694 specific binding represented 28 and 50% of total radioligand binding, respectively. These data in bovine and rat brain tissues indicate that bovine area postrema can be used with 3H-GR65630, 3H-BRL43694, or 3H-LY278584 for drug screening or molecular investigations of the brain 5HT3 receptor: only 3H-LY278584 can be used for studies on the regulation and/or the molecular properties of 5HT3 receptors in rat cortical homogenates. 5HT3 receptors were solubilized from bovine area postrema and characterized using 3H-GR65630.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates.

The temporal relationship between the alpha-thrombin-induced increase in transendothelial permeability and the alpha-thrombin-mediated changes in several key transmembrane signaling events was examined in confluent monolayers of bovine pulmonary artery endothelial cells (BPAEC). The time courses of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] generation, changes in cytosolic [Ca2+] ([Ca2+]i), and reorganization of cytoskeletal F-actin were determined to assess the relationship between these events and the onset of the alpha-thrombin-induced increase in endothelial permeability. alpha-Thrombin (10(-7) M) increased the transendothelial 125I-albumin clearance rate half-maximally by approximately 1 min and maximally by approximately 2 min (160% over control level). The increase in permeability occurred concomitantly with reorganization of F-actin cytoskeleton (i.e., loss of peripheral band and increased stress fiber density) and increased actin polymerization. Stimulation of fura-2-loaded BPAEC with 10(-7) M alpha-thrombin produced a typical biphasic rise in [Ca2+]i. The initial rapid increase in [Ca2+]i peaked by approximately 16 s after thrombin challenge and the [Ca2+]i response showed a slow decrease to half-maximal within 50 s. The alpha-thrombin-induced increase in permeability as well as the increase in [Ca2+]i were consistently preceded by increased Ins(1,4,5)P3 generation detectable within 10 s after thrombin challenge. These results indicate that alpha-thrombin triggers a cascade of events (i.e., Ins(1,4,5)P3 generation and the ensuing rise in [Ca2+]i), which may comprise the second messengers that mediate F-actin reorganization and the increase in endothelial permeability.

Actins↗
PubMed1992

Follicle-stimulating hormone increases guanine nucleotide-binding regulatory protein subunit alpha i-3 mRNA but decreases alpha i-1 and alpha i-2 mRNA in Sertoli cells.

FSH interacts with a guanine nucleotide-binding protein (G-protein)-coupled receptor, which in turn modulates signal transduction via the G-protein subunit alpha s. However, it is unknown whether FSH regulates alpha-subunit gene expression and whether G-protein alpha-subunit genes other than alpha s are modulated in FSH-stimulated signal transduction. Regulation of mRNA for alpha s and alpha i-2 was studied in primary cultures of rat Sertoli cells because these proteins are linked to the control of adenylyl cyclase. In addition, mRNA for alpha i-1 and alpha i-3 were quantified because these proteins are putatively linked to ion channels but have not been well characterized in the Sertoli cell. Northern blot analyses demonstrated that FSH induced a dose-dependent increase in steady state levels of alpha i-3 mRNA. In contrast, FSH caused a dose-dependent decrease in levels of alpha i-1 and alpha i-2 mRNA. No significant effect of FSH on alpha s mRNA levels was detectable. The time course of FSH effects showed a 75% decrease in alpha i-1 mRNA levels, a 50% decrease in alpha i-2 mRNA levels and a nearly 3-fold increase in levels of alpha i-3 mRNA between 4-6 h of treatment with 100 ng/ml FSH. Steady state levels of alpha i-1 and alpha i-2 mRNA returned to pretreatment levels after 10 h FSH treatment, while alpha i-3 mRNA returned to a new steady state level approximately 50% greater than the pretreatment level.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

NAN-190: agonist and antagonist interactions with brain 5-HT1A receptors.

NAN-190 has been reported to be a 5-HT1A antagonist in drug discrimination studies. In order to determine if the effect of NAN-190 was directly due to competitive inhibition at 5-HT1A receptors, 5-HT1A-mediated inhibition of adenylyl cyclase in hippocampal membranes was investigated. NAN-190 (10(-10)-10(-5) M), by itself, was found to have no effect on forskolin-stimulated adenylyl cyclase. NAN-190, however, did shift the 5-carboxamidotryptamine (a 5-HT1A agonist) log-concentration inhibition curve to the right in a concentration-dependent manner, typical of competitive antagonism. Schild analysis revealed a KB of 1.9 nM for NAN-190. Thus, NAN-190 appeared to be a potent competitive 5-HT1A antagonist using the in vitro adenylyl cyclase system. [3H]NAN-190 was synthesized and its 5-HT1A receptor binding properties were characterized and compared with the 5-HT1A agonist radioligand, [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT). The 5-HT1A agonists, serotonin (5-HT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) competed with equal affinities regardless of the radioligand used to label the 5-HT1A receptors. [3H]NAN-190 and [3H]8-OH-DPAT labeled the same number of sites in rat hippocampus, striatum and frontal cortex. Guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) and 5-guanylyl-imidodiphosphate (GppNHp), non-hydrolyzable analogs of GTP, inhibited specific [3H]NAN-190 binding. Adenosine-5'-O-(3-thio)triphosphate (ATP gamma S) and 5-adenylyl-imidodiphosphate (AppNHp) were ineffective. This guanylyl nucleotide-specific effect is generally associated with agonist radioligand binding to a GTP-binding protein coupled receptor. However, [3H]8-OH-DPAT was far more sensitive than [3H]NAN-190 to the Bmax reducing effects of GTP and GTp gamma S. We propose that the test for a reduction in Bmax by non-hydrolyzable guanylyl nucleotides may be more sensitive than other tests for quantifying agonist activity and may demonstrate that NAN-190 has low intrinsic activity. In summary, NAN-190 displayed antagonist-like properties in functional models of 5-HT1A receptor activity and possibly partial agonist-like properties in radioligand binding experiments.

Adenylyl Cyclases↗
PubMed1988

Guanosine diphosphate binding, metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes.

Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of [3H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a Ka of 4.5.10(6) M-1 and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5'-O-[3-thio]triphosphate (Gppp[S]), was most effective as an inhibitor of [3H]GDP binding (ED50 = 4.8.10(-8) M), whereas guanosine 5'-O-[2-thio]diphosphate (Gpp[S]) was less potent (ED50 = 3.4.10(-7) M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of Gppp[S], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg2+ concentration with EDTA and including App[NH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that Gpp[S] inhibited (P less than 0.05) Gppp[S]-stimulated adenylate cyclase activity (Ki = 1.8.10(-7) M), whereas basal activity remained unaffected. Addition of Gpp[S] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a Koff value of 6 +/- 1.min-1. Using a two-stage pre-incubation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound Gpp[S] for Gppp[S] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides.

Adenylyl Cyclases↗
PubMed1987

Testicular compensatory hypertrophy in the hemicastrated calf: effects of exogenous estradiol.

Unilaterally orchidectomized (hemicastrated) bull calves were studied to monitor possible changes in serum concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the phase of testicular compensatory growth, to examine the characteristics of LH and FSH binding to the testis of the post-pubertal animal, and to determine whether any of these responses were altered by exogenous estradiol. Twenty-four calves were assigned randomly at one week of age to a 2 X 2 factorial experiment involving intact control (I) and hemicastrated animals (H), as well as estradiol-implanted intact (I+E2) and hemicastrated animals (H+E2). Relative to I, testis growth was accelerated in H and suppressed in I+E2 and H+E2. Mean testis weights at 27 weeks of age were 42 +/- 4, 72 +/- 6, 12 +/- 1 and 14 +/- 1 g for the four respective treatment groups. Serum FSH, but not serum LH, was positively associated with the accelerated testis growth of H. LH and FSH binding per testis were both enhanced approximately twofold in the testis from hemicastrated animals relative to those from intact calves. In contrast, estradiol markedly suppressed the number of LH-binding sites per testis in both I and H calves, but only suppressed the number of FSH-binding sites per testis in H calves. LH-affinity constants were not affected by treatment, whereas those for FSH were significantly decreased by estradiol. In conclusion, neonatal hemicastration results in elevated serum FSH, testicular compensatory hypertrophy, and an increased number of gonadotropin receptors in the bovine testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Guanine triphosphate-binding site regulation by follicle-stimulating hormone and guanine diphosphate in membranes from immature rat Sertoli cells.

GTP binding to Sertoli cell membranes has been investigated using [3H]5'-guanylyl-beta gamma-imidodiphosphate [[3H]Gpp(NH)p], a nonhydrolyzable analog of GTP. Binding of [3H]Gpp(NH)p Gpp(NH)p to membranes prepared from Sertoli cells in serum-free culture was proportional to membrane protein concentration in the range of 5-50 micrograms. Competitive displacement studies using adenine (ATP, ADP, and AMP) and guanine nucleotides [GTP, GDP, GMP, and Gpp(NH)p] indicated that only GTP, its analog Gpp(NH)p, and GDP were effective ligands. The relative potencies were Gpp(NH)p much greater than GTP greater than GDP, as characterized by ED50 values of 0.8, 2.5, and 4 microM, respectively. Competitive inhibition by GTP, however, was similar to that by Gpp(NH)p in the presence of a nucleoside triphosphate-regenerating system, suggesting the involvement of an active GTPase. Equilibrium binding studies indicated a single high affinity site for GTP with a Ka of 3.3 +/- 0.2 X 10(7) M-1. This value was supported by other studies in which an association rate constant of 1.8 X 10(6) M-1 min-1 and a dissociation rate constant of 2.4 X 10(-2) min-1 were estimated. Maximal binding of [3H]Gpp(NH)p to Sertoli cell membranes ranged from 30-55 pmol/mg protein. FSH enhanced [3H]Gpp(NH)p binding by about 50% (P less than 0.05), reflecting an increase in the number of available binding sites rather than an effect on Ka. When GDP was preincubated with membranes in the absence of FSH, the number of available binding sites for [3H]Gpp(NH)p was decreased. This reduction in available binding sites by pretreatment with GDP could be reversed by adding FSH during the equilibrium binding analysis. These studies have demonstrated specific high affinity binding of Gpp(NH)p to Sertoli cell membranes with an affinity comparable to that required for activation of FSH-sensitive adenylate cyclase. Furthermore, a potent GTPase activity associated with the Sertoli cell membrane is responsible for rapid hydrolysis of GTP to GDP and may participate in inactivation of GTP-dependent adenylate cyclase activity. The role of FSH in the regulation of nucleoside binding appears to be in facilitating exchange of GTP for GDP by enhancing the release of bound GDP.

Animals↗
PubMed1984

Cellular processing of follicle-stimulating hormone by Sertoli cells in serum-free culture.

Specific binding of radioiodinated human follicle-stimulating hormone [( 125I]hFSH) to receptors on the cell surface of immature rat Sertoli cells in serum-free culture (4-7 days) was concentration-, time- and temperature-dependent with an apparent steady state of binding stained after 2 h at 4, 18 or 32 degrees C. Analysis of [125I]hFSH binding data indicated an apparent Ka of 3 X 10(9) M-1 with approximately 670 fmoles binding sites/mg protein. The radioiodinated hFSH (60-70 microCi/micrograms) bound to receptor and stimulated estradiol production, thus confirming its biological activity and Sertoli cell viability. Approximately 50% of the specifically bound radioactivity was lost from the cell surface after 0.25 h at 32 degrees C, 1 h at 18 degrees C or 4 h at 4 degrees C. Most (greater than 90%) of the receptor-bound hormone was removed from the cell surface and degraded to [125I]mono- and diiodotyrosine within 4 h at 32 degrees C. FSH degradation was inhibited at low temperatures (4 degrees C) and by the lysosomotropic agents chloroquine, NH4Cl or concanavalin A. Bacitracin and methylamine, inhibitors of internalization, were effective in blocking degradation but not loss of membrane-bound [125I]hFSH. Thus it appears that, as with small peptide and simple protein hormones, complex glycoprotein hormone-receptor complexes, such as FSH Sertoli cell receptors, are endocytosed by normal target cells and subsequently degraded, most probably within the lysosomes.

Animals↗
PubMed1983

Inhibition of 125I-human follicle-stimulating hormone binding to receptor by a low molecular weight fraction of bovine follicular fluid: inhibitor concentration is related to biochemical parameters of follicular development.

Pools of follicular fluid (FF) were obtained from large or small follicles of cows which were pregnant or in the luteal phase of the estrous cycle. Cells present in each FF pool were collected by centrifugation and measured for content of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors. Steroid levels in FF were quantitated by radioimmunoassay (RIA). Since the quantity of bovine follicular cells (mostly granulosa cells) was limited, FSH binding inhibition was studied utilizing a calf testis receptor system. Low (less than 6000) molecular weight (Mr) fractions prepared by dialysis were shown to account for most (76 to 94%) of the FSH binding inhibition (FSH-BI) present in unfractionated FF. The concentration of low Mr FSH-BI was higher in pools of FF from cows in the luteal phase of the estrous cycle than in pools of FF from pregnant cows. The concentration of low Mr FSH-BI was also higher in FF pooled from small follicles than in FF pooled from large follicles of either pregnant or luteal phase cows. Relative concentrations of receptors for gonadotropins (FSH, LH) on granulosa cells were used to rank the pools according to relative degree of follicular maturation. Other parameters of follicular maturation were concentration of estrogens and the ratio of estrogens to androgens in FF. Biochemical parameters for follicular atresia were the concentration of androgens and the ratio of estrogens to androgens in FF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Inhibition of FSH action on granulosa cells by low molecular weight components of follicular fluid.

Immature female rats were injected with a single dose (10 IU) of pregnant mare serum gonadotropin to induce growth and maturation of ovarian follicles. Using such ovaries as a model, we tested the effects of low molecular weight subfractions of charcoal-absorbed bovine follicular fluid (FF-c) on (a) radioiodinated human FSH (125I-hFSH) binding to ovarian homogenates, (b) ovine FSH-stimulated adenylate cyclase activity in granulosa cell homogenates and (c) cAMP production by intact granulosa cells. The follicular fluid was fractionated by ultrafiltration through membranes of differing pore-sized into molecular weight components of 1000-5000 (passing Amicon H1P-5 hollow fibers but retained by Amicon UM-2 membrane) and 500-1000 (passing Amicon UM-2 membrane but retained by Amicon Um-05 membrane). These low molecular weight fractions inhibited 125I-hFSH binding to ovarian receptors, FSH-stimulated cAMP production by rat granulosa cells and FSH-stimulated, as well as fluoride-ion-stimulated adenylate cyclase activity in granulosa cell homogenates. Inhibition of FSH-stimulated adenylate cyclase activity by the FF subfractions was non-competitive as determined by double reciprocal plot analysis. Our results suggest that modulation of FSH effects on granulosa cells may be mediated by low molecular weight constituents of follicular fluid.

Adenylyl Cyclase Inhibitors↗
PubMed1982

Biochemical properties of the testicular follitropin-receptor system.

The interaction of FSH with membrane receptors from rat and calf testis has been studied in some detail. The FSH receptor has been solubilized through use of the nonionic detergent Triton X-100 and highly purified by affinity chromatography on Affigel-10 coupled to ovine FSH. Hormone binding activity of the solubilized receptor has been preserved for extended periods through use of the structure-stabilizing agent glycerol. Other components of the FSH testes receptor system including the guanyl nucleotide binding protein and adenylate cyclase have been solubilized by nonionic detergents and also found to be stabilized by glycerol. FSH binding activity has been observed in testes cytosol and represents a putative class of receptors prepared from testes in the absence of detergent. The concentration of this buffer-soluble component decreased with age and increased concomitantly with loss of membrane receptors consequent to their down-regulation after administration of exogenous FSH. Phospholipids seem involved in the interaction of FSH with membrane-bound, detergent-solubilized, and buffer-soluble FSH binding activity. Phospholipids may maintain or stabilize a particular receptor conformation necessary for interaction with the hormone. A specific role for GTP seems indicated in regulation of FSH-stimulated adenylate cyclase activity in immature rat testis. Follitropin binding to testes receptor appears modulated by a variety of factors present in serum, testes extracts, follicular fluid, and seminal plasma, which are poorly understood at present. Inhibition of FSH binding by seminal plasma best-fit by a model proposing two hormone binding sites per receptor molecule, where binding to one site decreases the affinity of the other site for FSH. As a result of studies in this and other laboratories, the molecular endocrinology of FSH interaction with testis receptors is becoming increasingly understood.

Adenylyl Cyclases↗