Climate and weather factors affecting winter sheltering by shoreline Copper Rockfish Sebastes caurinus in Howe Sound, British Columbia.

We monitored winter sheltering behavior of Copper Rockfish (Sebastes caurinus) in layered boulders at a shoreline in British Columbia and identified possible links to climate change and evolutionary adaptation. During late autumn and winter, these fish were inside the interstices of the boulder pile (termed "winter sheltering"); these fish were actively swimming above the boulders during spring through early fall. Sheltering duration did not vary between normal and most El Niño years (154-177 days). Sheltering longer than 6 months occurred during strong La Niña winters (197-241 days). Additionally, the proximate stimulus for entry into sheltering was intense Arctic outflow windstorms. Emergence from sheltering appears linked to water temperatures, occasionally related more to spring river flooding (snowmelt). The winter sheltering behavior we describe may be unique to shoreline populations in inland seas. Sheltering may confer a fitness advantage by conserving energy or reducing mortality from predation, thus increasing longevity and chances for successful reproduction. Our observations suggest that an ONI threshold of 0.8 °C or greater would be better suited than the current 0.5 °C threshold used to define ONI events.

GABA transporter heterogeneity: pharmacology and cellular localization.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. GABA is cleared from the synaptic cleft by specific, high-affinity, sodium- and chloride-dependent transporters, which are thought to be located on presynaptic terminals and surrounding glial cells. While early studies suggested a distinction between neuronal and glial GABA transport, molecular cloning has revealed the existence of genes for four distinct GABA transporters (termed GAT-1, GAT-2, GAT-3 and BGT-1), thus revealing a greater heterogeneity than previously suspected. Heterologous expression has allowed a detailed characterization of their pharmacological properties, and has revealed that GAT-1 is the site of action of the anticonvulsant drug, Tiagabine. In-situ hybridization and immunocytochemistry demonstrate that each transporter has a unique regional distribution in the brain; in conjunction with experiments utilizing cell cultures, the neuronal vs glial localization of the various transporters is being elucidated. Future studies will be directed at determining the role of each transporter in the regulation of GABAergic transmission, and in the design of additional subtype-specific inhibitors, which may serve as novel therapeutic agents for the treatment of neuropsychiatric disorders.

Localization of messenger RNAs encoding three GABA transporters in rat brain: an in situ hybridization study.

Localization of the messenger RNAs encoding three gamma-aminobutyric acid (GABA) transporters, termed GAT-1, GAT-2, and GAT-3, has been carried out in rat brain using radiolabeled oligonucleotide probes and in situ hybridization histochemistry. Hybridization signals for GAT-1 mRNA were observed over many regions of the rat brain, including the retina, olfactory bulb, neocortex, ventral pallidum, hippocampus, and cerebellum. At the microscopic level, this signal appeared to be restricted to neuronal profiles, and the overall distribution of GAT-1 mRNA closely paralleled that seen in other studies with antibodies to GABA. Areas containing hybridization signals for GAT-3 mRNA included the retina, olfactory bulb, subfornical organ, hypothalamus, midline thalamus, and brainstem. In some regions, the hybridization signal for GAT-3 seemed to be preferentially distributed over glial cells, although hybridization signals were also observed over neurons, particularly in the retina and olfactory bulb. Notably, hybridization signal for GAT-3 mRNA was absent from the neocortex and cerebellar cortex, and was very weak in the hippocampus. In contrast to the parenchymal localization obtained for GAT-1 and GAT-3 mRNAs, hybridization signals for GAT-2 mRNA were found only over the leptomeninges (pia and arachnoid). The differential distribution of the three GABA transporters described here suggests that while each plays a role in GABA uptake, they do so via distinct cellular populations.

Cloning and expression of a betaine/GABA transporter from human brain.

A cDNA clone encoding a human gamma-aminobutyric acid (GABA) transporter has been isolated from a brain cDNA library, and its functional properties have been examined in mammalian cells. The nucleotide sequence predicts a transporter with 614 amino acids and 12 putative transmembrane domains. The highest degree of amino acid identity is with a betaine/GABA transporter originally cloned from the dog termed BGT-1 (91%) and a related transporter from mouse brain (87%). These identities are similar to those for species homologues of other neurotransmitter transporters and suggest that the new clone represents the human homologue of BGT-1. The transporter displays high affinity for GABA (IC50 of 30 microM) and is also sensitive to phloretin, L-2,4-diaminobutyric acid, and hypotaurine (IC50 values of approximately 150-400 microM). The osmolyte betaine is approximately 25-fold weaker than GABA, displaying an IC50 of approximately 1 mM. The relative potencies of these inhibitors at human BGT-1 differ from those of mouse and dog BGT-1. Northern blot analysis reveals that BGT-1 mRNA is widely distributed throughout the human brain. The cloning of the human homologue of BGT-1 will further our understanding of the roles of GABA and betaine in neural function.

Molecular cloning of an orphan transporter. A new member of the neurotransmitter transporter family.

A complementary DNA clone predicted to encode a novel transporter was isolated from rat brain and the localization of its mRNA was examined. The cDNA, designated rB21a, predicts a protein with 12 putative transmembrane domains that exhibits significant sequence homology with neurotransmitter transporters. Expression studies have not yet identified the endogenous substrate for this transporter, but the presence of rB21a mRNA within the leptomeninges of the brain suggests the transporter may regulate CSF levels of its substrate. The cloning of rB21a provides the means to determine its physiological functions and the potential to design novel, transporter-based therapeutic agents for neurological and psychiatric disorders.

Re-evaluation of GABA transport in neuronal and glial cell cultures: correlation of pharmacology and mRNA localization.

Molecular cloning has revealed the existence of four distinct transporters for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), termed GAT-1, GAT-2, GAT-3, and BGT-1. To determine which of the cloned transporters are in neurons and which are in glia, we have undertaken a combined pharmacological and molecular biological study using cell cultures derived from rat brain. In neuronal cultures approximately 70% of GABA transport is sensitive to the GAT-1-selective ligand NNC-711 and drug potencies at this site correlate well with their potencies at GAT-1; GAT-1 mRNA is abundant in these cultures as determined by northern blot analysis. Drug potencies at the NNC-711-resistant component correlate well with their potencies at GAT-2 and GAT-3, whose pharmacological profiles are similar to one another. Northern blots reveal the presence of mRNA for GAT-3 in neuronal cultures, whereas GAT-2 and BGT-1 mRNAs are not detected. Type 1 astrocyte cultures exhibit very low levels of GABA transport activity, which has very low potency for GABA but high potency for taurine. Such cultures have mRNA for a taurine transporter and BGT-1, but not for GAT-1, GAT-2, and GAT-3. In cultures containing O-2A progenitor cells and Type 2 astrocytes, approximately 75% of GABA uptake is sensitive to NNC-711 and drug potencies at this site correlate well with their potencies at GAT-1; GAT-1 mRNA is abundant. Drug potencies at the NNC-711-resistant component correlate well with their potencies at GAT-2 and GAT-3; mRNAs for both of these transporters are present (though GAT-2 mRNA is the more abundant), as is BGT-1 mRNA. In summary, these data demonstrate heterogeneity of both neuronal and glial GABA transport.

Tiagabine, SK&F 89976-A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The synaptic action of GABA is terminated by rapid uptake into presynaptic terminals and surrounding glial cells. Molecular cloning has revealed the existence of four distinct GABA transporters termed GAT-1, GAT-2, GAT-3, and BGT-1. Pharmacological inhibition of transport provides a mechanism for increasing GABA-ergic transmission, which may be useful in the treatment of various neuropsychiatric disorders. Recently, a number of lipophilic GABA transport inhibitors have been designed and synthesized, which are capable of crossing the blood brain barrier, and which display anticonvulsive activity. We have now determined the potency of four of these compounds, SK&F 89976-A (N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid), tiagabine ((R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3- piperidencarboxylic acid), CI-966 ([1-[2-[bis 4-(trifluoromethyl)phenyl]methoxy]ethyl]-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid), and NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,4,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride), at each of the four cloned GABA transporters, and find them to be highly selective for GAT-1. These data suggest that the anticonvulsant activity of these compounds is mediated via inhibition of uptake by GAT-1.

Design, synthesis and evaluation of substituted triarylnipecotic acid derivatives as GABA uptake inhibitors: identification of a ligand with moderate affinity and selectivity for the cloned human GABA transporter GAT-3.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Molecular biology has revealed the presence of four high-affinity GABA transporters in the brain, GAT-1, GAT-2, GAT-3, and BGT-1, the latter transporting both GABA and the osmolyte Betaine. We have shown that known GABA uptake inhibitors such as SK&F 89976-A, CI-966, and Tiagabine exhibit high affinity and selectivity for GAT-1. In the present paper we describe the design and synthesis of a novel series of triarylnipecotic acid derivatives for evaluation as GABA uptake inhibitors. The design lead for this series of compounds was the nonselective GABA uptake inhibitor EGYT-3886, [(-)-2-phenyl-2-[(dimethylamino)ethoxy]-(1R)- 1,7,7-trimethylbicyclo[2.2.1]heptane]. From this series of compounds (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic+ ++ (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic+ ++ acid, 4(S) was identified as a novel ligand with selectivity for GAT-3. 4(S) displayed an IC50 of 5 microM at GAT-3, 21 microM at GAT-2, > 200 microM at GAT-1, and 140 microM at BGT-1. This compound will be an important tool for evaluating the role of GAT-3 in neural function.

Cloning of the human homologue of the GABA transporter GAT-3 and identification of a novel inhibitor with selectivity for this site.

Molecular cloning has revealed the presence of four high-affinity GABA transporters in the brain. The existence of three of these sites, GAT-2, GAT-3, and BGT-1, was unknown prior to their cloning and almost nothing is known of the role they play in regulating GABAergic transmission. In large measure our paucity of knowledge is attributable to the lack of specific inhibitors for these sites. In the present communication we describe the cloning and expression of the human homologue of GAT-3, and the identification of an inhibitor, (S)-SNAP-5114, with selectivity for this site. (S)-SNAP-5114 displays an IC50 of 5 microM at GAT-3, 21 microM at GAT-2, and > or = 100 microM at GAT-1 and BGT-1. Due to its lipophilicity, (S)-SNAP-5114 is also expected to cross the blood-brain-barrier and therefore, should be an important tool for evaluating the role of GAT-3 in neural function.

Cell-specific coupling of the cloned human 5-HT1F receptor to multiple signal transduction pathways.

We recently described the cloning of a fifth member of the 5-hydroxytryptamine (5-HT)1 (serotonin1) receptor class that inhibits adenylyl cyclase, namely the human 5-HT1F receptor (Adham et al. 1993a). In the present study we have examined in greater detail the functional coupling of the 5-HT1F receptor in two different cell lines, NIH-3T3 and LM(tk-) fibroblasts (receptor densities of 1.7 and 4.4 pmol/mg protein, respectively). The maximal inhibitory response elicited by 5-HT was significantly greater in NIH-3T3 as compared to LM(tk-) cells, whereas the EC50 values were comparable. To investigate the relationship between receptor occupancy and inhibition of cAMP accumulation mediated by 5-HT1F receptors in NIH-3T3 cells (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The half-maximal response required only about 10% receptor occupancy, consistent with a receptor reserve of 90% (88 +/- 2.1%, n = 4) for 5-HT-induced inhibition of FSCA. Despite the presence of such a high degree of receptor reserve, a range of intrinsic activities was displayed by structurally diverse classes of compounds. For example, sumatriptan and lysergol were as efficacious as 5-HT itself and thus acted as full agonists, whereas metergoline and 1-NP behaved as partial agonists and as shown previously (Adham et al. 1993a), methiothepin was a silent antagonist (Kb = 438 nM). We have also investigated activation of additional signal transduction pathways by the 5-HT1F receptor and found that the responses differ in the two cell lines with respect to stimulation of phospholipase C. For example, in NIH-3T3 cells no elevation of inositol phosphates (IP) of [Ca2+]i was observed even at very high agonist concentrations (100 microM). In contrast, in LM(tk-) cells concentrations of 5-HT as low as 10 nM induced stimulation of IP and a rapid increase of [Ca2+]i. The 5-HT1F receptor failed to alter arachidonic acid release in either cell line. The maximal increase in IP accumulation in LM(tk-) cells was modest, averaging about 100% above basal. The increases of IP and [Ca2+]i required 5-HT concentrations less than one order of magnitude greater than those inhibiting FSCA (EC50 = 17, 55 and 8 nM, respectively), and both responses were blocked by 100 microM methiothepin. All three responses (cAMP, IP, and [Ca2+]i) were sensitive to pertussis toxin pre-treatment, suggesting the involvement of Gi/Go protein(s) in these signal transduction pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

Dual coupling of cloned human 5-hydroxytryptamine1D alpha and 5-hydroxytryptamine1D beta receptors stably expressed in murine fibroblasts: inhibition of adenylate cyclase and elevation of intracellular calcium concentrations via pertussis toxin-sensitive G protein(s).

The second messenger coupling of cloned human 5-hydroxytryptamine (5-HT)1D alpha and 5-HT1D beta receptors stably expressed in murine fibroblasts (LM (tk-)) was investigated. Clonal cell lines expressing similar receptor densities (Bmax = 750-950 fmol/mg) were used in this study. 5-HT (EC50 = 1.5-2.0 nM) and sumatriptan (EC50 = 6-14 nM), a selective 5-HT1D agonist, produced dose-dependent inhibition of forskolin-stimulated cAMP accumulation in intact cells transfected with the 5-HT1D alpha or 5-HT1D beta receptor gene. The maximal inhibitory responses elicited by these agonists were slightly greater with the 5-HT1D alpha receptor (approximately 90%) than the 5-HT1D beta receptor (approximately 80%). 5-HT (EC50 = 1.7-2.4 nM) and sumatriptan (EC50 = 8-18 nM) also evoked dose-dependent elevations in intracellular calcium concentrations ([Ca2+]i), with EC50 values that were indistinguishable from those for inhibition of forskolin-stimulated cAMP accumulation. Cells expressing 5-HT1D beta receptors displayed significantly larger 5-HT-induced increases in [Ca2+]i than did cells expressing 5-HT1D alpha receptors (206 nM versus 114 nm increase; p < 0.01). Dose-dependent elevations in inositol phosphates (IP) were also observed after application of 5-HT (EC50 = 29-54 nM) or sumatriptan (EC50 = 73-481 nM); the maximal increases in IP accumulation were modest (51-69%) for both 5-HT1D subtypes. In contrast to the cAMP and calcium responses, the concentration-response curves for IP accumulation were shifted to the right at least 10-fold. Methiothepin, a nonselective 5-HT1 antagonist, competitively antagonized the cAMP response, yielding an apparent dissociation constant (Kb) of 3-4 nM for the 5-HT1D receptors. Methiothepin (10 microM) significantly reduced the elevations in [Ca2+]i (> 90%) and IP (> 75%) evoked by saturating concentrations (1 microM) of agonists. All three functional responses were significantly attenuated (> 90%) by pretreatment with 100 ng/ml pertussis toxin. The sumatriptan-induced elevation of [Ca2+]i via activation of the 5-HT1D subtypes may provide a molecular mechanism of action by which sumatriptan could directly constrict cerebral blood vessels and alleviate migraine symptoms.

Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain.

cDNA clones encoding two novel gamma-aminobutyric acid (GABA) transporters (designated GAT-2 and GAT-3) have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The transporters display high affinity for GABA (Km approximately 10 microM) and exhibit pharmacological properties distinct from the previously cloned neuronal GABA transporter (GAT-1). Both transporters require sodium and chloride for transport activity. The nucleotide sequences of GAT-2 and GAT-3 predict proteins of 602 and 627 amino acids, respectively, which can be modeled with 12 transmembrane domains, similar to the topology proposed for other cloned neurotransmitter transporters. Localization studies indicate that both transporters are present in brain and retina, while GAT-2 is also present in peripheral tissues. The cloning of these transporter genes from rat brain reveals previously undescribed heterogeneity in GABA transporters.

Cloning and expression of a high affinity taurine transporter from rat brain.

A cDNA clone (designated rB16a) encoding a taurine transporter has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The nucleotide sequence of the clone predicts a 621-amino acid protein with significant homology to other neurotransmitter transporters. Hydropathy analysis reveals stretches of hydrophobic amino acids indicative of 12 transmembrane domains, similar to those proposed for other cloned neurotransmitter transporters. The transporter displays high affinity for taurine (Km approximately 40 microM) and is dependent on external sodium and chloride for transport activity. Specific transport is sensitive to inhibition by beta-alanine and gamma-aminobutyric acid, similar to taurine transporters in vivo. Localization studies demonstrate that the transporter mRNA is located in both the brain and peripheral tissues. The structural similarity of the taurine transporter to neurotransmitter transporters is consistent with a neuromodulatory role for taurine in the nervous system.

Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors.

A complementary DNA clone encoding a transporter for glycine has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The transporter displays high affinity for glycine (KM approximately 100 microM) and is dependent on external Na+ and Cl-. Northern blot analysis indicates that the distribution of the mRNA encoding the glycine transporter is restricted to the nervous system. In situ hybridization data are consistent with roles for the transporter in both glycine neurotransmission and glycine modulation of N-methyl-D-aspartate (NMDA) receptors in the hippocampus. The identification of this transporter therefore opens the study of the molecular mechanisms underlying both inhibitory glycinergic transmission and NMDA-mediated excitatory transmission.

Resting [Ca2+]i and [Ca2+]i transients are similar in fibroblasts from normal and Alzheimer's donors.

Previous studies have reported that resting concentrations of intracellular calcium ion were markedly reduced in cultured dermal fibroblasts from Alzheimer's disease patients and that the ability of these cells to respond to serum stimulation was also decreased as compared to both young and age-matched control cells. In this study we have carefully reexamined these parameters in several of the same lines of fibroblasts and fail to find major differences in resting cytosolic calcium [Ca2+]i, in the response of [Ca2+]i to serum stimulation or in cell spreading in the AD cells as compared to young controls. The present findings suggest that cytoplasmic ionic calcium levels are neither pathognomonic for Alzheimer's cells nor of diagnostic value.

Nerve growth factor potentiates bradykinin-induced calcium influx and release in PC12 cells.

To investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin-induced calcium increases in PC12 cells. Short-term (1 h) treatment with NGF increased the potency of bradykinin to raise intracellular calcium by about 10-fold, whereas long-term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100-fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short-term potentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long-term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC12 cells was unaltered by short-term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol 1,4,5-trisphosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself.

Flunitrazepam photoaffinity labeling of the GABA(A) receptor reduces inhibition of [3H]Ro15-4513 binding by GABA.

The benzodiazepine drugs modulate gamma-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA(A) receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [3H]Ro15-4513, [3H]flumazenil (Ro15-1788), and [3H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [3H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]Ro15-4513. The increase in [3H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Kd, with no change in Bmax. Following photoaffinity labeling, negative modulation of [3H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [3H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [3H]Ro15-4513.

Acidification-dependent dissociation of endocytosed insulin precedes that of endocytosed proteins bearing the mannose 6-phosphate recognition marker.

A key step in the sorting of endocytosed ligands from their receptors is dissociation, which is triggered by the acidic pH of endosomes. To determine whether dissociation occurs synchronously for all ligands, we compared in Chinese hamster ovary cells the intracellular dissociation of insulin, which dissociates between pH 6.3 and 7.0, with that of lysosomal hydrolases bearing the mannose 6-phosphate recognition marker (Man-6-P proteins), which dissociate around pH 5.8. Chinese hamster ovary cells were pulsed for 2 min with 125I-insulin, acid-washed to remove surface binding, and chased. During a 40-min period, about 50% of the internalized 125I-insulin was released intact via a retrocytotic pathway. Retrocytosis was not inhibited by monensin, suggesting that the release was not dependent on acidic endosomes. The remaining insulin dissociated from its receptor in an acidification-sensitive manner and was eventually degraded. Dissociation was 70% complete within 5 min of internalization. When cells were similarly incubated with 125I-Man-6-P proteins, about 35% of the internalized radioactivity was released during a 1-h chase, reflecting proteolytic maturation of the Man-6-P proteins. Dissociation of Man-6-P proteins was acidification-dependent (i.e. inhibited by monensin), and was 50% complete after about 11 min. The results indicate that acidification-dependent dissociation of ligands does not occur in a single step and suggest that multiple endocytic compartments are involved in receptor/ligand sorting.

Kinetics of alpha 2-macroglobulin endocytosis and degradation in mutant and wild-type Chinese hamster ovary cells.

The production of Chinese hamster ovary (CHO) cell mutants which are defective in endocytosis has led to a greater understanding of the process by which cells sort ligands and their receptors. Robbins and coworkers have obtained CHO mutants which are resistant to diphtheria toxin, defective in the delivery of endocytosed lysosomal enzymes to lysosomes, and have a decreased uptake of iron from transferrin (Robbins et al.: J. Cell Biol. 96:1064-1071, 1983). We have previously shown that these CHO mutants are markedly deficient in the acidification of early endocytic compartments (Yamashiro and Maxfield: J. Cell Biol. 105:2713-2721, 1987). In this study we examined the endocytosis of alpha 2-macroglobulin (alpha 2M) to determine whether the defects in early endosome acidification would alter the processing of this ligand. We found that the CHO mutants DTG 1-5-4 and DTF 1-5-1 bind, internalize, and degrade 125I-alpha 2M in a manner similar to the wild-type cells. We also found that the CHO mutants retain the ability to recycle the receptors for alpha 2M. Since the binding of alpha 2M is greatly reduced at mildly acidic pH (approximately 6.8), only slight acidification of the endosomal compartment should be sufficient to achieve sorting of alpha 2M from its receptor. In contrast, lysosomal enzymes require more acidic conditions (pH less than 6.0) for dissociation. The different behavior of the two ligands provides biochemical evidence for a partial (but not complete) defect in early endosome acidification in the mutants. The data also indicate that pH regulation in a relatively narrow range can achieve differential sorting of various ligands.