Phosphorylation Of Membrane Proteins Research Articles

Proton Transport and Phosphorylation of Tonoplast Polypeptides from Zucchini Are Stimulated by the Phospholipid Platelet-Activating Factor

The ether phospholipid platelet-activating factor and certain similar phospholipids, including lysophosphatidylcholine, are known to stimulate both H(+) transport and protein phosphorylation in plant microsomal membranes. In the present work, several polypeptides in highly purified tonoplast membranes from zucchini (Cucurbita pepo L.) showed platelet-activating factor-dependent phosphorylation. Comparison of protein phosphorylation in different membrane fractions separated by sucrose step density gradient centrifugation indicated that some of the phosphoproteins were contaminants or were common to several membrane fractions, but platelet-activating factor-dependent phosphorylation of peptides at 30, 53, and perhaps 100 kilodaltons was tonoplast specific. The phosphoprotein of 53 kilodaltons was shown by three different approaches (one- and two-dimensional polyacrylamide gel electrophoresis, western blots, and immunoprecipitation) to cross-react with antibody raised against the B subunit of the tonoplast ATPase from red beet (Beta vulgaris L.).

Calcium-dependent protein phosphorylation in morning glory hypocotyls

The membrane fraction isolated from hypocotyls of morning glory showed protein kinase activity, which catalysed phosphorylation of serine and threonine residues in the membrane proteins. The activity was stimulated by 0.1-1 μM Ca 2+. Addition of calmodulin could not cause further stimulation, but reduced Ca 2+ -dependent protein kinase activity. The activity was restored by a calmodulin antagonist. The effect of calmodulin was not observed in phosphorylation of erogenous substrates by the protein kinase, suggesting that the function of an increased Ca 2+ -calmodulin complex may be to depress the phosphorylation of membrane proteins.

Enhanced expression of membrane phosphoproteins tyrosine phosphorylation in estrogen-induced kidney tumors

We demonstrate for the first time that the expression of tyrosine containing membrane phosphoproteins is elevated in estrogen-induced kidney tumors, which is evident from both the types of experiments, i.e., alkali-resistant phosphorylation of membrane proteins and immunoprecipitation of tyrosine containing phosphoproteins. Tyrosine phosphorylation of proteins or peptides was modulated by the growth factors (EGF, IGF-I) and by the inhibitors of tyrosine protein kinase(s). The kinetic analyses revealed that tumor membranes have high affinity and catalytically more efficient tyrosine phosphorylating kinase enzyme(s) compared to that of normal membranes which have low affinity and catalytically less efficient kinase enzyme(s). It is proposed that overexpression of tyrosine containing membranal phosphoproteins may be involved in the induction and growth of estrogen-induced renal neoplasm.

Regulation of Ca 2+ uptake in skeletal muscle by 1,25-dihydroxyvitamin D 3: Role of phosphorylation and calmodulin

Experiments were carried out to obtain information about the mechanism underlaying the fast action of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) in skeletal muscle. N-2'- o-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP), similarly as 1,25(OH) 2D 3 (5 × 10- −10 M), rapidly increased 45Ca uptake by soleus muscle from vitamin D-deficient chicks (+25% and +98% at 3 min and 10 min, respectively) in a dose-dependent manner. The effects of the cAMP analog (10 μM) and 1,25(OH) 2D 3 could be abolished by the Ca 2+-channel blocker nifedipine and the calmodulin antagonist flufenazine. Calmodulin binding by two muscle microsomal proteins of 28 kDa and 30 kDa was stimulated within l min of exposure of the tissue to 1,25(OH) 2D 3. Direct effects of the sterol on membrane calmodulin binding were shown with isolated microsomes. The l,25(OH) 2D 3-mediated rise of [ 125I]calmodulin binding to microsomal membranes was dependent on the presence of medium ATP. Forskolin (10 μ M) and cAMP (10 μM) also increased [ 125I]calmodulin binding (+75% and +64%, respectively, with respect to controls). Pretreatment of microsomal membranes with cAMP-dependent protein kinase inhibitor (1 μ/ml) or addition of alkaline phosphates (1 U/ml) after hormonal treatment caused complete inhibition of l,25(OH) 2D 3-induced [ 125I]calmodulin binding to microsomal membrane proteins. These results imply modifications of membrane protein phosphorylation through the cAMP signal pathway and in turn of calmodulin binding in the mechanism by which 1,25(OH) 2D 3 rapidly stimulates skeletal muscle Ca 2+ uptake.

Modulation of 1,25-dihydroxyvitamin D3-dependent Ca2+ uptake in skeletal muscle by protein kinase C.

In vitro studies have shown that short exposure (1-10 min) of vitamin D-deficient chick soleus muscle to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] causes an acute stimulation of tissue 45Ca uptake through voltage-gated Ca2+ channels, with parallel increases in cyclic AMP levels, adenylate cyclase activity and membrane protein phosphorylation. We further investigated the involvement of protein kinases in the rapid effects of 1,25(OH)2D3 on skeletal muscle. The hormone was found to stimulate the protein kinase C (PKC) activity of muscle membranes. The PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nM) was found to rapidly stimulate muscle 45Ca uptake, mimicking 1,25(OH)2D3. Increases of 68% and 46% were observed at 1 and 15 min of exposure to PMA respectively. The effects of PMA were dose-dependent (50-200 nM) and were specific, since the inactive analogue 4 alpha-phorbol was without effect. Analogously to the effects of the sterol, PMA-enhanced 45Ca uptake was abolished by the Ca2+ channel antagonists nifedipine (30 microM) and verapamil (50 microM). Staurosporine (10 nM), a PKC inhibitor, surprisingly potentiated 1,25(OH)2D3-dependent stimulation of 45Ca uptake. Exposure of skeletal muscle to PMA (100 nM) plus 1,25(OH)2D3 (1 nM) produced a less pronounced effect on 45Ca uptake than either agent alone. PMA also decreased muscle cyclic AMP levels. These results suggest a regulatory link between the two major transmembrane signalling systems in the mechanism of action of 1,25(OH)2D3 in skeletal muscle.

Growth factor-dependent phosphorylation of membrane proteins in cultured human retinal pigment epithelial cells.

Recently, growth factors are known to phosphorylate tyrosine residues of proteins to regulate cellular functions. We investigated growth factor-dependent phosphorylation of membrane proteins in cultured human retinal pigment epithelial (RPE) cells. The phosphorylation experiments were done in membrane preparations of cultured RPE cell and the reaction was started by applying [32P]adenosine 5'-triphosphate (ATP) at 0 degrees C, and terminated after 0, 1, 5, 15, and 30 min. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were analyzed by autoradiography. Many proteins showed time-dependent phosphorylation. Among them, a 170 kDa protein showed platelet-derived growth factor (PDGF)-specific phosphorylation with both time- (up to 30 min) and dose-(maximal effect at 50 ng/ml) dependence. On the other hand, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed no specific phosphorylation. Phosphoaminoacids of the 170 kDa protein were analyzed by thin layer chromatography and autoradiography. Phosphotyrosine showed much higher radioactivity than phosphoserine or phosphothreonine. Consequently, PDGF induced phosphorylation of the 170 kDa protein which mainly consisted of phosphotyrosine. The data suggest that tyrosine phosphorylation of membrane protein is involved in signal transduction of PDGF in human RPE cells.

Enhanced membrane protein kinase C activity in myocardial ischemia.

The protein kinase activity in cytosol was similar in control, ischemic, and reperfused hearts; however, a 1.5-fold increase in membrane protein kinase activity was induced by ischemia and reperfusion. The H-7 inhibitable cytosolic protein kinase activity decreased by 40% with 30 min ischemia, while that of membrane fraction increased 1.8-fold. However, the CGS9343B inhibitable protein kinase activity in cytosolic fractions was unaffected by ischemia, while that of membrane increased by about 1.7-fold. These results suggest that myocardial ischemia is associated with enhanced protein kinase C and calmodulin-dependent kinase activities in membrane fraction. Furthermore, the results also suggest a translocation of protein kinase C activity from the cytosol to the membrane. Reperfusion of ischemic myocardium did not result in any further increase of protein kinase C and calmodulin-dependent kinase activities in the membrane. These enhanced protein kinase activities also resulted in an enhanced phosphorylation of endogenous membrane proteins. The creatine kinase released from the heart was increased by both ischemia and reperfusion. Therefore, these results suggest that biochemical cascades of reactions caused by enhanced membrane protein kinase C and calmodulin-dependent kinase activities may contribute to ischemic-reperfusion injury.

Endogenous phosphorylation and dephosphorylation of rat liver plasma membrane proteins, suggesting a 18 kDa phosphoprotein as a potential substrate for alkaline phosphatase

Purified rat liver plasma membranes were incubated for 0-60 min with [gamma-32P]ATP and analysis of 32P-labeled proteins by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the presence of two shifted kinetic phenomena. The use of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinases, allowed the identification of one as the endogenous protein phosphorylation. The other was shown to be the labeling of two phospho-intermediate forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1.], which have apparent molecular masses of 151 and 135 kDa. Bromolevamisole, a potent inhibitor of the enzyme, stabilized these phospho-intermediates, and consequent on this inhibition the labelling of a 18 kDa phosphoprotein was augmented. So, when alkaline phosphatase was studied in its native plasma membrane environment, a specificity of this enzyme over the endogenous phosphoproteins was established.

The Phospholipid Platelet-activating Factor Stimulates Proton Extrusion in Cultured Soybean Cells and Protein Phosphorylation and ATPase Activity in Plasma Membranes

Summary Medium acidification by cell cultures from soybean ( Glycine max. L.) was stimulated by the ether phospholipid platelet-activating factor (PAF), 1-O-alkyl-2-acetyl- sn -glycero-3-phosphocholine. Acidification was dependent on the concentration of PAF, on the cell density and on the presence of calcium ions in the medium. Ethyleneglycol-bis(aminoethylether)-N,N,N〲,N〲-tetraacetic acid (EGTA) equaling the calcium concentration reduced the proton extrusion whereas addition of the calcium ionophore A 23187 increased it. Cells in the logarithmic growth phase 3—4 days after inoculation, showed the strongest response to the lipid. In vitro PAF had a stimulatory effect on the ATPase activity in both the tonoplast-and the plasma membrane-enriched fraction. In microsomes isolated from cultured soybean cells PAF stimulated the phosphorylation of a 55 kDa, a 97 kDa and a 120 kDa polypeptide. With an antibody prepared against a cytosolic fragment of the plasma membrane H + -ATPase an immunostaining signal was obtained at 120 kDa, suggesting that the phosphoprotein having the same molecular mass could be the plasma membrane H + -ATPase. After membrane fractionation by a sucrose gradient the 55 kDa and the 97 kDa phosphoproteins were found predominantly in fractions of low density correlating with markers of either tonoplast or endoplasmatic reticulum.

Erythropoietin induces cytosolic protein phosphorylation and dephosphorylation in erythroid cells.

Erythropoietin, the prime regulator of red blood cell growth and differentiation, causes rapid changes in the phosphorylation of several integral plasma membrane proteins (Choi, H-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936; Choi, H-S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J. (1990) J. Biol. Chem. 265, 4143-4148). In the present study we have demonstrated that erythropoietin's signal is transduced rapidly to the cytosol resulting in specific phosphorylation/dephosphorylation events. Erythropoietin treatment of Rauscher murine erythroleukemia cells previously labeled with [32P]orthophosphate results in a rapid increase in phosphorylation of two cytosolic proteins, designated pp96 and pp80, and a decrease in phosphorylation of another protein, designated pp90. The relative molecular mass and pI of pp80 are virtually identical to those reported for the protein kinase C substrate p80, or "MARCKS protein." Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate also increases pp80 but not pp96 phosphorylation, suggesting that erythropoietin triggers a protein kinase C-dependent pathway to pp80 and a protein kinase C-independent pathway to pp96. The effect of erythropoietin on pp96 phosphorylation was also shown in nontransformed erythroid cells isolated from the spleens of phenylhydrazine-treated mice. In contrast, almost no 32P labeling of pp80 or pp90 was detected, and pp80 and pp90 protein were nearly absent from these normal cells. These differences in expression and phosphorylation of erythropoietin-sensitive phosphoproteins may be related to the growth factor independence or dependence of the erythroid cells.

Evidence on the participation of the 3',5'-cyclic AMP pathway in the non-genomic action of 1,25-dihydroxy-vitamin D 3 in cardiac muscle

Several studies have suggested that vitamin D plays a role in cardiovascular function. It has been recently shown that in vitro treatment of vitamin D-deficient chick cardiac muscle with physiological concentrations of 1,25-dihydroxy-vitamin D 3 (1,25(OH) 2D 3) induces a rapid (1–10 min) increase of tissue 45Ca uptake which can be suppressed by Ca channel blockers. The hormone simultaneously stimulated heart microsomal membrane protein phosphorylation. Experiments were performed to investigate the existence of a relationship between these changes and to obtain information about the mechanism involved in 1,25(OH) 2D 3-induced modifications in cardiac protein phosphorylation. Dibutyryl cyclic AMP (10 μM) and forskolin (10 μM), known activators of the cAMP pathway, produced time courses of changes in 45Ca uptake by chick heart tissue similar to 1,25(OH) 2D 3 (10 −10 M). Analogously to the hormone, the effects of both compounds were abolished by nifedipine (30 μM) and verapamil (10 μM). In agreement with these observations, 1,25(OH) 2D 3 significantly increased (34–70%) heart muscle cAMP levels within 1–10 min of treatment. In addition, 1,25(OH) 2D 3 and forskolin caused similar changes in cardiac microsomal membrane protein phosphorylation (e.g. stimulation in 43 kDa and 55 kDa proteins). These changes were also evidenced by direct exposure of isolated heart microsomes to 1,25(OH) 2D 3, suggesting a direct membrane action of the hormone. The fast effects of 1,25(OH) 2D 3 on dihydropyridine-sensitive cardiac muscle Ca uptake could be reproduced in primary-cultured myocytes isolated from chick embryonic heart. Furthermore, the effects of the hormone could be suppressed by a specific protein kinase A inhibitor. These results suggest that 1,25(OH) 2D 3 affects heart cell calcium metabolism through regulation of Ca channel activity mediated by the cAMP pathway.

Insulin-stimulated tyrosine phosphorylation of a 43 kDa protein in rat liver membranes

The insulin receptor (IR) tyrosine kinase is essential for the regulation of different cellular functions by insulin. This may occur by a direct phosphorylation of membrane and/or cytoplasmic proteins by the IR tyrosine kinase. Hence it is important to identify putative physiological substrates for the IR tyrosine kinase. In this study we found that the glycoprotein fraction from rat liver membranes contain a 43 kDa protein (pp43) which, like the beta-subunit of IR, is phosphorylated in an insulin-dependent manner. A 25-fold enhancement of 32P incorporation into pp43 by insulin was found under optimal conditions. Half-maximal phosphorylation of pp43 and the beta-subunit of IR were attained at 66 nM and 60 nM insulin, respectively. Mn2+ (Ka = 1.0 mM) was much better than Mg2+ (Ka = 6.3 mM) in supporting pp43 phosphorylation. Insulin-stimulated phosphorylation of pp43 (t1/2 = 3.6 min) proceeded at a much slower rate compared to that of the beta-subunit of IR (t1/2 = 1.2 min). Phosphoamino acid analysis of pp43 revealed that both tyrosine and serine are phosphorylated in the ratio 4:1. Tyrosine, but not serine, phosphorylation was increased 12-fold by insulin. Phosphorylation of pp43 occurred on 4 major tryptic peptides. Comparison to the tryptic phosphopeptides from IR beta-subunit suggest that pp43 was not derived from IR beta-subunit by proteolysis. Our results suggest that pp43 may be an endogenous substrate for the IR tyrosine kinase.

Candidate proteins for conductive chloride transport in porcine ileal brush-border membrane.

Conductive transport of chloride ion is important in controlling ion and fluid secretion by exocrine tissues. The current study was directed at identifying proteins in the intestinal brush-border membrane that may be involved with conductive chloride transport. Reaction of total brush-border membrane protein with phenyl-isothiocyanate inhibited conductive chloride transport into brush-border membrane vesicles. The conductive transport process was protected from this inhibition by including the ligands Cl- and alpha-phenylcinnamate in the reaction mixture. Brush-border membrane protein protected by this procedure and labeled with fluorescein had an apparent molecular mass in the region of 130 and 23 kDa on separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation of brush-border membrane protein with [gamma-32P] ATP and endogenous protein kinase under conditions causing activation of chloride conductance in membrane vesicles caused the transfer of 32P to several proteins, including ones in the same molecular size range (130 and 23 kDa) as those identified by the fluorescein labeling procedure. Conductive chloride transport in porcine intestinal brush-border vesicles may occur via proteins identified by this differential labeling procedure.

Calretinin, a neuronal calcium binding protein, inhibits phosphorylation of a 39 kDa synaptic membrane protein from rat brain cerebral cortex

The neuronal calcium binding protein calretinin was studied for possible effects on brain protein phosphorylation. Calretinin (100 nM) inhibited the appearance of a calcium stimulated 39 kDa phosphoprotein within a synaptic membrane fraction following sucrose density centrifugation. Calmodulin or a specific protein kinase C inhibitor had no effect on either the phosphorylation of the 39 kDa protein or the inhibition produced by calretinin. At the same concentration, calretinin produced a slight increase in the phosphorylation of several other synaptic membrane proteins which appeared additive with the stimulation produced by either calmodulin or phosphatidylserine in the presence of calcium.

Deficient protein kinase C activity in turnip, a Drosophila learning mutant

The Drosophila mutant turnip was initially isolated based on poor learning performance (Quinn, W.G., Sziber, P.P., and Booker, R. (1979) Nature 277, 212-214). Here we show that turnip is dramatically reduced in protein kinase C (PKC) activity. In addition, turnip flies are deficient in phosphorylation of a 76-kDa head membrane protein (hereafter pp76) which is a major substrate for protein kinase C in homogenates of wild-type flies. Reduced PKC activity, defective pp76 phosphorylation, and most of turnip's learning deficiency co-map genetically to a region on the X-chromosome, 18A5-18D1-2, spanned by the deletion Df(1)JA27. Apparently turnip+ is not a structural gene for PKC because Drosophila PKC genes map elsewhere in the genome. Our results suggest that turnip gene product is required for activation of PKC and that PKC plays a role in associative learning in Drosophila.

Correction of erythrocyte abnormalities in idiopathic calcium-oxalate nephrolithiasis and reduction of urinary oxalate by oral glycosaminoglycans

Calcium-oxalate nephrolithiasis is associated with a defect in erythrocyte oxalate self-exchange and an abnormal rate of erythrocyte membrane protein phosphorylation. There is evidence that glycosaminoglycans (GAGs) have a regulatory effect on both of these processes. This study tested the hypothesis that modifications of erythrocyte oxalate self-exchange induced by oral GAGs are paralelled by similar changes in overall oxalate metabolism. 40 patients with idiopathic calcium-oxalate nephrolithiasis were treated for 15 days with 60 mg/day of a mixture of GAGs. By day 15 of treatment there were significant reductions from baseline in erythrocyte oxalate self-exchange (mean [SD] 16·7 [1·18] vs 2·59 [1·63] × 102 per min; p<0·005) and erythrocyte membrane protein phosphorylation (55·8 [7·3] vs 72·9 [6·8] × 10-3 cpm/mg protein; p<0·005), but also in urinary oxalate excretion (0·24 [0·09] vs 0·31 [0·15] mmol/24 h; p < 0·005). This finding suggests similar changes in both erythrocytes and other cells more important in oxalate handling. The changes had reversed by 15 days after withdrawal of treatment. Acute intravenous administration of GAGs (60 mg) induced a fall in carbon-14-labelled oxalate renal clearance (143 [13] vs 169 [28] ml/min; p < 0·005), which strongly suggests the participation of the kidney. However, reduced oxalate absorption from the intestine, and even decreased synthesis of oxalate, cannot be ruled out.

The use of excitation fluorescence spectroscopy in the study of short-term chloroplast membrane reorganisation

The advantages of the use of excitation fluorescence spectroscopy in the investigation of PS I absorption cross-section dynamics during phosphorylation of membrane proteins or heat treatment have been shown. After phosphorylation in the light, there occurred only 15–20% enhancement in absorption cross-section of F735 — PS I peripheral antenna — at the expense of chlorophyll b and short wavelength chlorophyll a species in the region 674–676 mm, which is attributed to the light-harvesting pigment complex of Photosystem II. Phosphorylation in the dark with dithionite caused increases of 30–35% in PS I absorption cross-section at the expense of Chl b, Chl a absorbing at 674–676 mm and long-wavelength PS II internal antenna chlorophyll at 685–687 nm. The same changes in the region of 685–687 mm were found after heat treatment of chloroplasts. The application of excitation fluorescence spectroscopy made it possible to estimate directly for the first time not only the extent of changes in absorption cross-section of PS I but also to describe which complex of PS II is responsible.

Light-dependent phosphorylation of Photosystem II polypeptides maintains electron transport at high light intensity: separation from effects of phosphorylation of LHC-II

Phosphorylation of chloroplast thylakoid membrane proteins, including LHC-II and PS II polypeptides D1, D2, CP43 and the psbH gene product, occurs under plastoquinone-reducing conditions. Dephosphorylation of LHC-II and PS II phosphoproteins proceeds under plastoquinone-oxidizing conditions, but with differing kinetics. This study shows that phosphorylation sites of PS II polypeptides are substrates for exogenous alkaline phosphatase activity, whereas those of LHC-II are not. By combined use of exogenous and endogenous phosphatase activities four types of thylakoid membrane are generated, with (i) both LHC-II and PS II polypeptides phosphorylated, (ii) only PS II polypeptides phosphorylated, (iii) only LHC-II phosphorylated and (iv) neither class of polypeptide phosphorylated. Examination of PS II electron transport in each membrane type shows that a mechanism involving phosphorylation of PS II polypeptides specifically is required for PS II function at high light intensity. Phosphorylation of LHC-II results in an inhibition of PS II electron transport at limiting light intensity which can therefore be attributed to decreased PS II absorption cross-section. QB binding is unaffected by phosphorylation in any membrane type, nor are changes in function at the donor side of PS II involved. The role of PS II polypeptide phosphorylation in energy dissipation and protection from photoinhibition is discussed.

Altered erythrocyte protein kinase C activity and membrane protein phosphorylation in chronic myelogenous leukemia

the membrane protein kinase C (PKC) content was found to be higher in erythrocytes form patients suffering from chronic myelogenous leukemia (CML) compared to normal erythrocytes. PKC activity was also higher in the cytosol and after translocation to the membrane, as assessed by histone phosphorylation. The increased PKC activity in CML erythrocytes was associated with abnormal phosphorylation of protein 4.1. Since phosphorylation-dephosphorylation mechanisms are likely candidates for controlling membrane protein associations, the altered PKC activity may be one of the factors responsible for altered thermal sensitivity and mechanical stability of CML erythrocytes.

Regulation of ANF receptor internalization: Involvement of extracellular calcium

Receptor mediated internalization of 125I-ANF (99–126) and the underlying mechanism was studied in PC12 cells. Phosphorylation of PC12 cell plasma membrane proteins at 0°C or 37°C was not altered in presence of ANF (99–126) or c-ANF (4–23). Exposure of cells to phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) did not alter the endocytic rate or extent of 125I-ANF (99–126) internalization. When cells were treated with a combination of PMA and the calcium ionophore A23187, internalization was not stimulated. Incubation with A23187 (10 μM) alone decreased 125I-ANF (99–126) internalization by 22% in Ca 2+ containing medium. Cell surface binding increased 10% in the presence of Ca 2+ compared to Ca 2+ free medium, irrespective of the presence of A23187. Ca 2+ appears to play an important role in the binding of ANF to the receptor and initiation of ligand-receptor complex internalization. Activation of protein kinase C or receptor phosphorylation is not an essential step in initiating ANF receptor internalization.