Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10).

Heterotrimeric G proteins are involved in the transduction of hormonal and sensory signals across plasma membranes of eukaryotic cells. Hence, they are a critical point of control for a variety of agents that modulate cellular function. Activation of these proteins is dependent on GTP binding to their alpha (Galpha) subunits. Regulators of G protein signaling (RGS) bind specifically to activated Galpha proteins, potentiating the intrinsic GTPase activity of the Galpha proteins and thus expediting the termination of Galpha signaling. Although there are several points in most G protein controlled signaling pathways that are affected by reversible covalent modification, little evidence has been shown addressing whether or not the functions of RGS proteins are themselves regulated by such modifications. We report in this study the acute functional regulation of RGS10 thru the specific and inducible phosphorylation of RGS10 protein at serine 168 by cAMP-dependent kinase A. This phosphorylation nullifies the RGS10 activity at the plasma membrane, which controls the G protein-dependent activation of the inwardly rectifying potassium channel. Surprisingly, the phosphorylation-mediated attenuation of RGS10 activity was not manifested in an alteration of its ability to accelerate GTPase activity of Galpha. Rather, the phosphorylation event correlates with translocation of RGS10 from the plasma membrane and cytosol into the nucleus.

Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vitro, radioreceptor activity and immunoactivity.

To establish whether sialic acid content is responsible for an observed 7-8-fold variability in bioactivity in vitro of highly purified human pituitary luteinizing hormone (hLH) isoforms, the bioactivity in vitro, radioreceptor activity and immunoactivity of hLH isoforms were determined before and after enzymatic desialylation. Three immunofluorometric assays with different hLH specificities allowed characterization of 13-24 pituitary hLH isoform preparations of pI 7.03-8.98 in terms of sialic acid content (1-5 sialic acid residues per LH molecule), bioactivity in vitro (4030-30,000 I.U. mg[-1]), radioreceptor activity (6420-25,400 I.U. mg[-1]) and hLH immunoactivity (2900-4400 to 18,300-27,300 I.U. mg[-1]). Significant positive correlations between sialic acid content and either immunoactivity or in vitro bioactivity were observed, whereas radioreceptor activity showed a curvilinear response. Following more than 90% removal of sialic acid, both in vitro bioactivity and radioreceptor activity were increased, although specific activity still differed between isoforms; immunoactivities were unaffected. It is concluded that the presence of the sialic acid residue(s) on hLH isoforms does partially contribute to the in vitro bioactivity and radioreceptor activity of the isoforms, but that hLH immunoactivity is independent of sialic acid content.

Structural and functional characterisation of hFSH and hLH isoforms.

Human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) are gonadotropins which are secreted as multiple forms by the pituitary. Evidence supporting the structural and functional heterogeneity of 15 purified hFSH isoforms and 20 purified hLH isoforms from pituitary extracts will be presented. Gonadotropin isoforms were purified by a combination of preparative isoelectric focusing and ion-exchange chromatography. The protein mass of each isoform was determined by amino acid analysis, which also correlated (data for hLH) (r = 0.999, P < 0.001, n = 15) with the UV area under the curve at 280 nm of the isoforms following gel-filtration HPLC. The alpha and beta subunits of FSH and LH were shown to be intact by SDS-PAGE under reducing condition, with no evidence of proteolytic nicking or presence of contaminating proteins. hFSH radioreceptor activity varied over a seven-fold range, and a positive correlation (r = 0.85, P < 0.001, n = 9) was observed between FSH receptor activity and the sialic acid (SA) content (1.5-13.7 mol SA/mol hFSH) of the isoforms, as determined by an HPLC-based microfluorometric assay. FSH in vitro activities varied over a similar range with a high correlation (r = 0.82, n = 15) with receptor activities, suggesting that the initial association of the hormone with the receptor is the key interaction with less differences attributed to subsequent effects in the signaling pathway. A similar result was seen with the hLH isoforms. To explore FSH/LH in vivo, the circulating half-life (LH/FSH) and the in vivo bioactivity (LH) using an acute in vivo assay was investigated. The clearance of hLH and hFSH showed a bi-exponential pattern for all isoform preparations with the proportion of the slower dissociating component (t 1/2 50-60 min) increasing three-fold with increasing sialic acid content of the isoform. The more rapidly cleared component (t 1/2 approx 10 min) is attributed to hepatically cleared gonadotropin, rather than gonadotropin equilibration between body compartments. The in vivo assay procedure for LH was based on the 24 h integrated plasma testosterone levels in rats following administration of graded doses of hLH isoform or standard. A 16-fold range in vivo activities between LH isoforms (n = 14) was observed. A comparison between hLH in vitro and in vivo activities showed a good correlation (r = 0.75) with the slope of the regression line (1.39) not significantly different from unity. These results suggest that in this acute in vivo assay method, the differences in circulating half-lives between hLH isoforms although large is not a key factor in their in vivo activity. However, in chronic in vivo assay systems the differences in clearance rates between isoforms may be important in their subsequent biological response. It is concluded that structural heterogeneity of FSH and LH contributes to functional differences, with a key interaction occurring at the receptor level. The contribution of sialic acid to these activities was also investigated.

In vivo bioactivities and clearance patterns of highly purified human luteinizing hormone isoforms.

Previous studies have shown that highly purified isoforms of human pituitary LH exhibited a 20-fold range of in vitro bioactivities. The aim of this study was to determine the corresponding plasma half-lives, metabolic clearance rates (MCR), and in vivo bioactivities of these human (h) LH isoforms. Cannulated adult male rats were administered hLH isoforms as a bolus i.v. injection. For the half-life studies, blood was then serially collected over a 6-h period, and serum was assayed for hLH using a specific immunofluorometric assay. All hLH (n = 19) isoforms exhibited biexponential disappearance profiles with an initial fast half-life (t 1/2) for component A of 12.8 +/- 3.7 min, followed by a slow component B with t 1/2 of 58.9 +/- 4.4 min. The prevalence of component B in relation to component A increased significantly (r = 0.81, P < 0.001) over a 3-fold range when correlated with the sialic acid content of the isoform. Similarly, the MCR showed a significant correlation (r = 0.77, P < 0.001) with sialic acid content. The basis for the two t 1/2 components was then investigated. In the first experiment, rat plasma containing primarily component B was collected 90 min after hLH isoform administration and injected into a second animal. Only component B was observed with no evidence of component A, which indicates that the two t 1/2 components are not the product of the redistribution of the hLH isoform between body compartments. In the second experiment, component B was found to be dependent on sialic acid content, as desialylated hLH isoforms showed a rapid disappearance (t 1/2 = 8.6 +/- 3.1) with the component B proportion decreasing to < 10% of that of the nondesialylated control. This data indicates that sialic acid protects component B from rapid clearance. In addition, the proportion of the two components is dependent on sialic acid content, suggesting that the molecular location of the sialic acid on the carbohydrate moieties of hLH has a critical role in the clearance process. To determine the in vivo bioactivity of the hLH isoforms, an acute in vivo bioassay was developed in male rats. The assay was based on the hLH dose-dependent increase in total testosterone release in the same rat model as used in the plasma disappearance studies. Using the second International Standard (IS) hLH (0.3 IU-2.6 IU/kg) as standard, a linear dose-response of 24-h integrated serum testosterone levels was observed, with an index of precision of 0.11. Using this in vivo assay, a 16-fold range in in vivo bioactivities (3,200 to 51,100 IU/mg) was observed for 14 hLH isoforms. These in vivo bioactivities correlated with sialic acid content (r = 0.78, P < 0.001), MCR (r = 0.56, P < 0.05) and LH in vitro bioactivity (r = 0.75, P < 0.001) as determined using mouse Leydig cells in culture. Desialylation lead to over a 100-fold decrease in in vivo bioactivity of hLH. It is concluded that hLH isoforms are cleared in vivo by a two-component clearance mechanism, the proportion of which varies between isoforms and is dependent on sialic acid content of the isoform. These findings suggest that the molecular location of sialic acid on the hLH isoform is critical in defining the plasma disappearance of component B, whereas the mechanism of elimination of component A may well involve the hepatic GalNAc-sulphate receptor. Using an in vivo bioassay, the 16-fold difference in bioactivity between isoforms is attributed primarily to differences in their in vitro activity at the cellular level with a minor influence (< 2-fold) due to differences in in vivo clearance.

Application of a sensitive HPLC-based fluorometric assay to determine the sialic acid content of human gonadotropin isoforms.

The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hLH) are glycoproteins which are microheterogeneous in terms of their charge and molecular size, as well as their in vitro and in vivo bioactivities. The aim of this study was to determine the contribution of variations in sialic acid (N-acetyl neuraminic acid) content to the structural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was released by partial acid hydrolysis (0.1 M TFA, 80 degrees C, 1 h) and derivatised with the fluorescent label DMB (1,2-diamino-4,5-methylenedioxybenzene) in conjunction with an internal standard (N-glycoyl-neuraminic acid). The derivatives were then separated by reversed-phase HPLC. This method allowed quantitation of the sialic acid content over a range of 5-100 pmol with between assay variation of < 6% for sialic acid released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparison of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromatography with pulsed amperometric detection yielded similar results, confirming the reliability of the fluorescence detection method. The sialic acid contents of 9 hFSH isoforms varied between 1.5-13.7 mol Neu5AC/mol FSH, whilst a range of 1.1-9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. The sialic acid content of the hFSH isoforms was also observed to be related to the hormonal specific activity in a radioreceptor assay, confirming that alterations in the carbohydrate structure can influence the FSH-receptor interaction. In contrast, the sialic acid content of the hLH isoforms was found to be not related to specific activity at the receptor level.

Immunological activities of highly purified isoforms of human FSH correlate with in vitro bioactivities.

In a recent study, a five- to eightfold range in human FSH radioreceptor activity (RRA) was documented for highly purified isoforms of FSH when the data were expressed on an FSH protein content basis as determined by amino acid analysis. This study examined the FSH in vitro bioactivity and immunoactivity of these preparations. FSH in vitro biological activity showed a five- to eightfold range in activity with a high correlation with the RRA values (r = 0.82). A similar five- to eightfold range of values was obtained with a specific FSH radioimmunoassay and an FSH two-site immunoassay with high correlations again observed between each other, between each immunoassay and with either the in vitro bioassay or the RRA method (r = 0.77-0.995). Although there was overall a close correlation between these assays, significant differences in ratios of activities between the in vitro bioassay and other methods were observed with highly purified FSH isoform preparations from different pI regions. The high correlation between in vitro bioassay/RRA methods and immunoassay methods over a wide range of isoform specific activities suggests that these methods are detecting similar structural features on each isoform. It is thus concluded that these immunoassays are not solely measuring hormone mass based entirely on amino acid composition. This conclusion raises questions about ratio measurements of FSH, where immunoassay methods are presumed to measure total protein content, and their application in physiological situations and clinical practice.

Isolation and characterization of human LH isoforms.

Thirty-nine human LH (hLH) isoforms were chromatographically separated from human pituitary extracts using a mild purification procedure which consisted of preparative isoelectric focusing, high-performance ion-exchange chromatography and immobilized metal-affinity chromatography. Twenty of these hLH isoforms were characterized by LH radioreceptor assay, SDS-PAGE and amino acid analysis, and 17 were shown to be highly purified (> 90% pure). The specific activities of these hLH isoforms ranged from 1980 to 38,650 IU/mg protein in terms of the 2nd IS for human pituitary LH, based on protein content as determined by amino acid analysis. hFSH and hTSH content were < 0.5% and < 7.8% respectively. The purity was assessed by silver staining on SDS-PAGE. Under non-reducing conditions, a single band of apparent molecular mass 23.5-24.5 kDa was observed, whereas under reducing conditions the isoforms migrated as two distinct bands, 21.1-22.4 kDa and 18.0-20.5 kDa, probably corresponding to the alpha and beta subunits of hLH respectively. The remaining three less pure isoform preparations (70-90% pure) contained additional bands of 16 kDa and 26.3 kDa under non-reducing conditions. All isoforms showed a low molecular mass band(s) of 11-14 kDa which was < 7% of stained material as assessed by densitometry. Amino acid composition of the 17 hLH isoforms was similar to the published cDNA composition of hLH. Further fractionation of one hLH isoform (hLH IIc) on reversed-phase high-performance liquid chromatography yielded four peaks identified by N-terminal sequencing as two alpha and two beta hLH subunits identical to their cDNA-derived N-terminal sequences. No additional sequences indicative of internal clipping of hLH were observed. The two pairs of alpha and beta subunits probably represent two separate hLH isoforms in this preparation. It was concluded that a mild purification procedure with high recoveries for the isolation of intact hLH isoforms has been developed, and 17 isoforms of high purity suitable for further biological and physicochemical characterization have been isolated. These isoform preparations are free of other contaminating proteins, but may still contain multiple hLH-related species.

Isolation and physicochemical characterization of human follicle-stimulating hormone isoforms.

Twenty hFSH isoforms were isolated from human pituitary extracts, 15 of which were highly pure. The mild purification procedure, which used a FSH RRA to monitor FSH activity, involved an initial fractionation of pituitary extracts by gel filtration and isoelectric focusing. Six pI regions (mean pI values, 3.63, 3.88, 4.07, 4.23, 4.84, and 5.13) of human (h) FSH were obtained and further fractionated on ion exchange and gel filtration HPLC. Recoveries of FSH radioreceptor activity at each stage were greater than 80%. Fifteen isoform preparations were judged as near homogeneous by HPLC-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibiting elution and migration behaviors consistent with the known mol wt of intact hFSH and its subunits. The remaining 5 isoform preparations contained a higher mol wt component that is probably hFSH related, as this component was detected after iodination, immunoprecipitation with hFSH antiserum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similar amino acid compositions were obtained for the 20 hFSH isoforms, except for evidence of some oxidative degradation of serine, threonine, and tyrosine and decreased levels of glutamic acid, possibly due to carboxy-terminal heterogeneity of the beta-subunit. An average amino acid composition value for all isoforms was comparable to that of 2 other highly purified hFSH preparations. Using the First International Standard for pituitary hFSH (83/575) as standard, radioreceptor activities were obtained ranging from 7,800-56,300 IU/mg protein. It is concluded that a mild purification procedure for the isolation of hFSH isoforms has been developed which gives high recoveries and has enabled the isolation of 15 isoforms in high purity suitable for further physicochemical and biological characterization.