Regulation of Nerve Growth Factor Action on Nsp100 Phosphorylation in PC12h Cells by Calcium

Previous work from these laboratories has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by treatment with nerve growth factor. This protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. The present studies have been designed to investigate the role of calcium in this action of nerve growth factor. It is shown here, using PC12h cells, that A23187, a calcium ionophore, and high levels of K+, a depolarizing stimulus, also decrease phosphorylation of Nsp100. Furthermore, the actions of nerve growth factor as well as those of A23187 and high levels of K+ are prevented by treatment of the cells with the calcium chelator EGTA. It is also shown that agents that raise levels of cyclic AMP in the cells, specifically dibutyryl cyclic AMP and cholera toxin, also decrease phosphorylation of Nsp100 but, in addition, increase phosphorylation of tyrosine hydroxylase. The action of these latter agents on Nsp100 is blocked by EGTA, but their action on tyrosine hydroxylase is not, indicating that even agents such as cholera toxin act on Nsp100 through a Ca2+-dependent mechanism.     

Identification of Nsp100 as elongation factor 2 (EF-2)

The nerve growth factor-sensitive phosphoprotein from PC12 cells, previously designated Nsp100, has been shown to be elongation factor 2 (EF-2). The criteria used for this identification include: (i) similarity of N-terminal sequence; (ii) phosphorylation by the same kinase; (iii) ADP-ribosylation mediated by diphtheria toxin; (iv) comparable function in cell-free protein synthesis. According to these criteria, Nsp100 and EF-2 are identical and the kinase that phosphorylates Nsp100 in PC12 cells is calcium/calmodulin kinase III.     

Fibroblast growth factor-induced decrease in the phosphorylation of Nsp100 mediated through a calcium-dependent mechanism and blocked by lectins

Separate treatment of PC12h cells with basic fibroblast growth factor (bFGF) and with epidermal growth factor (EGF) induced a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton soluble protein during phosphorylation with (gamma-32P)ATP of soluble extracts from the cells, as was seen previously with nerve growth factor (NGF). This 100,000-dalton soluble protein was designated in earlier studies as nerve growth factor-sensitive protein 100 (Nsp100). The inhibitory effects of bFGF and EGF on Nsp100 phosphorylation were prevented by pretreatment of PC12h cells with the calcium chelator, EGTA. Treatment of PC12h cells with the plant lectin wheat germ agglutinin (WGA), which binds to N-acetylglucosamine and sialic acid residues on glycoconjugates, blocked the inhibitory effects of bFGF, EGF, and NGF on Nsp100 phosphorylation. The blockage by WGA was reversed by the addition of the lectin-specific sugar N-acetylglucosamine to the PC12h cultures. Although pretreatment of PC12h cells with succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, failed to block the inhibitory effect of NGF on Nsp100 phosphorylation as described previously, it did prevent the inhibitory effect of bFGF on this phosphorylation. These data suggest that in PC12h cells bFGF and EGF induce a decrease in the phosphorylation of Nsp100 mediated through a Ca2(+)-dependent mechanism, as in the case of NGF. Furthermore, the blockage of the bFGF-induced inhibition of Nsp100 phosphorylation by WGA and its succinylated form indicates that N-acetylglucosamine residues of bFGF receptor molecules might be involved in the mechanism by which bFGF inhibits the phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Wheat germ agglutinin, concanavalin A, and lens culinalis agglutinin block the inhibitory effect of nerve growth factor on cell-free phosphorylation of Nsp100 in PC12h cells

It has been shown that in PC12 and its subclone PC12h treatment of the cells with nerve growth factor (NGF) induces a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton protein, designated in an earlier study as Nsp100, in the subsequent phosphorylation of soluble extracts from cells with (gamma-32P)ATP. In the present study, we show that plant lectins, wheat germ agglutinin (WGA), concanavalin A (Con A), and lens culinaris agglutinin (LCA), inhibit the action of NGF on Nsp100 phosphorylation in PC12h cells. Treatment of the cells with WGA, which binds to N-acetylglucosamine and sialic acid residues on glycoproteins, strongly blocked the inhibitory action of NGF on the protein phosphorylation. Con A and LCA, both of which recognize the same specific sugars (mannose, glucose), displayed only a moderate blocking effect. Unlike the native lectin, succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, and other lectins examined in this study did not inhibit the action of NGF on Nsp100. WGA-mediated inhibition of NGF action was reversed by the addition of N-acetylglucosamine and by the addition of a much lower concentration of a sialoglycoprotein, mucin, into the culture. Since the binding of succinylated WGA to N-acetylglucosamine residues of cell-surface glycoconjugates is not sufficient to prevent the action of NGF, WGA might act on sialic acid residues of the NGF receptor molecule to effect the inhibition of biological actions of NGF.     

S-100 Modulates Ca2+-Independent Phosphorylation of an Endogenous Protein (Mr= 19K) in Brain

Abstract: A new brain enzyme (tentatively named protein kinase X), which catalyzes protamine phosphorylation modulated by S-100, was reported recently. An endogenous substrate protein (M_r= 19K) for protein kinase × was isolated from brain by means of S-100-Sepharose 4B affinity chromatography. S-100, but not calmodulin, promoted phosphorylation of the 19K M_r protein in a Ca²⁺-independent manner, and this reaction was inhibited by gossypol. The substrate protein, localized in the particulate fraction, was present at a much higher level in brain from adult than neonatal rats (2-day-old), a developmental change similar to that seen for protein kinase X. It is suggested that a protein phosphorylation system modulated by S-100 exists in brain, and that this process may be involved in regulation of certain neural functions.     

Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells

New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.     

The salt-inducible kinase, SIK, is induced by depolarization in brain

Membrane depolarization of neurons is thought to lead to changes in gene expression that modulate neuronal plasticity. We used representational difference analysis to identify a group of cDNAs that are induced by membrane depolarization or by forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. One of these genes, SIK (salt-inducible kinase), is a member of the sucrose-nonfermenting 1 protein kinase/AMP-activated protein kinase protein kinase family that was also recently identified from the adrenal gland of rats treated with high-salt diets. SIK mRNA is induced up to eightfold in specific regions of the hippocampus and cortex in rats, following systemic kainic acid administration and seizure induction.     

S-100-Mediated Inhibition of Brain Protein Phosphorylation

The effects of the glial-specific, calcium-binding, S-100 protein on brain membrane and supernatant protein phosphorylation were assessed. S-100 concentrations as low as 5 micrograms/ml caused a marked inhibition of the phosphorylation of a soluble brain protein having a molecular weight of 73,000 daltons (73K). This protein was designated the S-100 protein-modulated phosphoprotein (SMP). Half-maximal inhibition of the phosphorylation of SMP by S-100 was obtained at concentrations of 12 micrograms/ml (0.57 microM). The inhibition of SMP phosphorylation by S-100 was calcium-dependent, with a calculated calcium Ka of 2.0 +/- 0.3 microM. SMP phosphorylation was also inhibited by calmodulin, but only partially and with a much lower potency. The inhibition of SMP phosphorylation by S-100 was not inhibited by fluphenazine, whereas the effect of calmodulin was. SMP was found in many brain areas, with the highest levels seen in the corpus callosum. Various peripheral tissues, such as kidney; liver; and pineal, pituitary, and adrenal glands, did not contain detectable SMP levels. At higher S-100 concentrations, greater than 10 micrograms/ml, the phosphorylation of several other soluble proteins was markedly inhibited. These proteins have molecular weights of 56K, 50K, and 47K. The phosphorylation of these proteins was enhanced by calmodulin. These data suggest that the S-100 protein may function to modulate the phosphorylation of brain proteins in a manner analogous to (although in a reciprocal fashion) that of calmodulin.     

Protein kinase c-β-like immunoreactivity in the developing and adult rat adrenal gland

Summary Protein kinase c--like immunoreactivity was studied in the adrenal gland of adult rats and at different pre- and postnatal stages of development (E17-P21) with an antibody specific to both the ₂₁ and - subtypes of the kinase. In the adult rat adrenal gland, the immunoreactivity was seen in numerous nerve fibres in the adrenal medulla both in bundles and individually forming occasionally dense networks around chromaffin cell groups. Several protein kinase c--immunoreactive fibres were also observed transversing the adrenal cortex towards the medulla. No remaining immunoreactive fibres two weeks after transection of the splanchnic nerve could be seen; nor was any immunoreactivity observed in the chromaffin cells of the adrenal medulla or in the cortical cells, but some faintly immunoreactive ganglion cells were detected in the adrenal medulla. The amount and distribution of protein kinase c--like immunoreactivity in the fetal and developing adrenals was very similar to that seen in the adrenal glands of adult rats. On the basis of its localization, the -subtype of protein kinase c does not appear to be directly involved in the release of catecholamines from the adrenal medulla, but it might have a role in the regulation of neurotransmitter release from preganglionic cholinergic neurons.     

IMMUNOHISTOCHEMICAL STUDIES OF S-100 PROTEIN DURING POSTNATAL ONTOGENESIS OF THE BRAIN OF TWO STRAINS OF RATS

Abstract— We have studied the dynamics of the appearance of cells reacting positively with anti-S-100 protein antiserum, during postnatal neurocytogenesis in the brain of rats of two strains differing in their susceptibility to sound stimuli. The postnatal time of appearance of cells reacting positively with anti-S-100 protein antiserum was somewhat later in rats susceptible to sound-induced seizures than in sound-resistant rats. These differences concerned mainly the cerebral cortex of 12-day-old rats. By day 21 of postnatal life these differences had disappeared. In subcortical structures of the brain, S-100 protein was first found on the 4th to the 5th day of life and the rate of appearance of cells containing this protein was similar in the two strains.     

THE EFFECT OF AGE, GROWTH RETARDATION AND ASPHYXIA ON ASCORBIC ACID CONCENTRATIONS IN DEVELOPING BRAIN

Abstract— The concentration of ascorbic acid in whole rat brain during the first week of postnatal life was up to 100 per cent higher than in adult animals. A progressive fall in concentration occurred between 4 and 30 days of age. Corresponding changes did not occur in liver and adrenal gland, two other organs rich in ascorbic acid. Rats subjected to growth retardation during the fetal and suckling periods had, at 25 days of age, levels of ascorbic acid in the cerebellum and brainstem significantly higher than those of control animals. A period of prolonged asphyxia in 5-day-old rats resulted in a significant 4 per cent reduction in whole brain ascorbic acid concentration.     

Lower rates of protein degradation in developing rat brain.

Protein-degradation rates in developing rat brain were estimated from the decay in total radioactivity in proteins labelled by a single intraperitoneal injection of NaH14CO3 to 5-day-old animals. In contrast with previous reports, our results indicate that degradation rates are lower in developing than in adult brain and suggest that in brain, as has been observed in liver, adrenal gland, muscle, cultured mammalian cells and bacteria, reduced rates of protein degradation contribute to the increase in protein content under conditions of rapid growth.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

Characterization of Insulin-Like Growth Factor-I Receptors in PC 12 Pheochromocytoma Cells and Bovine Adrenal Medulla

Competitive binding studies indicated that PC12 cells have receptors for insulin-like growth factor-I (IGF-I). There are approximately 11,000 +/- 1,500 IGF-I receptors/cell; these receptors have an apparent KD for IGF-I of 7.2 +/- 0.6 nM. Covalent cross-linking of 125I-IGF-I to PC12 cells labeled a 125,000-130,000-Mr protein, presumably the alpha-subunit of the IGF-I receptor. Although PC12 cells also have insulin receptors, the 125I-IGF-I appeared to be cross-linked to IGF-I receptors, because 100 nM IGF-I competed for labeling but 100 nM insulin did not. Bovine chromaffin cells also have IGF-I receptors. The protein tyrosyl kinase activity of IGF-I receptors from bovine adrenal medulla and PC12 cells was examined after purification of the receptors by wheat germ agglutinin-Sepharose chromatography. IGF-I (10 nM) stimulated autophosphorylation of the beta-subunits of the IGF-I receptors from both preparations; the beta-subunits from both sources had Mr values of approximately 97,000. IGF-I also stimulated phosphorylation of the synthetic substrate poly(Glu:Tyr)4:1 by both receptor preparations. IGF-I (IC50 of approximately 0.2 nM) was much more potent than insulin at stimulating phosphorylation of poly(Glu:Tyr) by the bovine adrenal medulla preparation. A maximal concentration of IGF-I (10 nM) increased phosphorylation approximately threefold. IGF-I was slightly more effective than insulin at stimulating the phosphorylation of poly(Glu:Tyr) by the PC12 cell receptor preparation, but neither ligand produced a maximal effect at concentrations up to 100 nM. This result probably reflects the presence of comparable numbers of IGF-I and insulin receptors on PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP-dependent protein kinase is not involved in the in vivo activation of tyrosine hydroxylase in the adrenal gland after decapitation

Tyrosine hydroxylase is activated in the adrenal gland in vivo after acute stresses, such as decapitation or electroconvulsive shock. In nonstressed animals that are anesthetized with pentobarbital prior to surgical removal of the adrenals, approximately 5-10% of the enzyme molecules are in the activated form, whereas in stressed animals, approximately 40-50% of the enzyme molecules are in the activated form. In the present study, we have tested the hypothesis that the stress-induced activation of the adrenal enzyme in vivo is due to the phosphorylation of the enzyme by cyclic AMP-dependent protein kinase. Soluble adrenal tyrosine hydroxylase prepared from either stressed or nonstressed rats is incubated in vitro with [gamma-32P]ATP and purified cyclic AMP-dependent protein kinase under optimal conditions for the phosphorylation of the enzyme. Using this assay, we have measured the number of vacant sites remaining on the enzyme, which are available for in vitro phosphorylation by cyclic AMP-dependent protein kinase. These studies suggest that the initial, in vitro rate of phosphorylation of tyrosine hydroxylase isolated from stressed rats is less than the initial rate of phosphorylation of the enzyme isolated from nonstressed rats. However, there is no significant difference in the final level of 32P phosphorylation of tyrosine hydroxylase isolated from either stressed or nonstressed rats. We conclude that, even though phosphorylation of tyrosine hydroxylase by cyclic AMP-dependent protein kinase leads to the activation of the enzyme under in vitro conditions, this mechanism cannot account for the activation of the enzyme in vivo in the adrenal gland following decapitation.     

A Nerve Growth Factor-Dependent Protein Kinase That Phosphorylates Microtubule-Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three- to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg²⁺ for activity but not Mn²⁺ or Ca²⁺. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF-dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.     

S100B increases proliferation in PC12 neuronal cells and reduces their responsiveness to nerve growth factor via Akt activation

S100B is a Ca2+-modulated protein of the EF-hand type expressed in high abundance in a restricted set of cell types including certain neuronal populations. S100B has been suggested to participate in cell cycle progression, and S100B levels are high in tumor cells, compared with normal parental cells. We expressed S100B in the neuronal cell line PC12, which normally does not express the protein, by the Tet-Off technique, and found the following: (i) proliferation was higher in S100B+ PC12 cells than in S100B- PC12 cells; (ii) nerve growth factor (NGF), which decreased the proliferation of S100B- PC12 cells, was less effective in the case of S100B+ PC12 cells; (iii) expression of S100B made PC12 cells resistant to the differentiating effect of NGF; and (iv) interruption of S100B expression did not result in an immediate restoration of PC12 cell sensitivity to the differentiating effect of NGF. Expression of S100B in PC12 cells resulted in activation of Akt; increased levels of p21WAF1, an inhibitor of cyclin-dependent kinase (cdk) 2 and a positive regulator of cdk4; increased p21WAF1-cyclin D1 complex formation; and increased phosphorylation of the retinoblastoma suppressor protein, Rb. These S100B-induced effects, as well as the reduced ability of S100B+ PC12 cells to respond to NGF, were dependent on Akt activation because they were remarkably reduced or abrogated in the presence of LY294002, an inhibitor of the Akt upstream kinase phosphatidylinositol 3-kinase. Thus, S100B might promote cell proliferation and interfere with NGF-induced PC12 cell differentiation by stimulating a p21WAF1/cyclin D1/cdk4/Rb/E2F pathway in an Akt-mediated manner.     

Mammalian myosin I alpha, I beta, and I gamma: new widely expressed genes of the myosin I family

A polymerase chain reaction strategy was devised to identify new members of the mammalian myosin I family of actin-based motors. Using cellular RNA from mouse granular neurons and PC12 cells, we have cloned and sequenced three 1.2-kb polymerase chain reaction products that correspond to novel mammalian myosin I genes designated MMI alpha, MMI beta, MMI gamma. The pattern of expression for each of the myosin I's is unique: messages are detected in diverse tissues including the brain, lung, kidney, liver, intestine, and adrenal gland. Overlapping clones representing full-length cDNAs for MMI alpha were obtained from mouse brain. These encode a 1,079 amino acid protein containing a myosin head, a domain with five calmodulin binding sites, and a positively charged COOH-terminal tail. In situ hybridization reveals that MMI alpha is highly expressed in virtually all neurons (but not glia) in the postnatal and adult mouse brain and in neuroblasts of the cerebellar external granular layer. Expression varies in different brain regions and undergoes developmental regulation. Myosin I's are present in diverse organisms from protozoa to vertebrates. This and the expression of three novel members of this family in brain and other mammalian tissues suggests that they may participate in critical and fundamental cellular processes.     

Localization of lipoprotein lipase mRNA in selected rat tissues

Measurements of enzymatic activity have demonstrated that lipoprotein lipase (LPL), the principal enzyme responsible for hydrolysis of circulating triglyceride, is present in a number of tissues including brain, kidney, and adrenal gland. To determine the sites of synthesis of LPL in these tissues, in situ hybridization studies were performed using a non-sense 35S-labeled RNA probe produced from a 624-bp mouse LPL cDNA fragment. Control studies were performed with a sense RNA strand. Using 5-10-micron sections of 5-day-old rat brain, strong hybridization was found in pyramidal neurons of the hippocampus. Positive hybridization, indicating the presence of LPL mRNA, was also found in brain cortex and in the intermediate lobe of adult rat pituitary gland. Specific areas of adrenal and kidney medulla showed hybridization with the probe. LPL mRNA is, therefore, present in a number of specific regions of the body. LPL in these areas may not be important in regulating circulating levels of lipoproteins, but may be essential for cellular uptake, binding, and transfer of free fatty acids or other lipophilic substances.