On the expansion of ribosomal proteins and RNAs in eukaryotes.

While the ribosome constitution is similar in all biota, there is a considerable increase in size of both ribosomal proteins (RPs) and RNAs in eukaryotes as compared to archaea and bacteria. This is pronounced in the large (60S) ribosomal subunit (LSU). In addition to enlargement (apparently maximized already in lower eukarya), the RP changes include increases in fraction, segregation and clustering of basic residues, and decrease in hydrophobicity. The acidic fraction is lower in eukaryote as compared to prokaryote RPs. In all eukaryote groups tested, the LSU RPs have significantly higher content of basic residues and homobasic segments than the SSU RPs. The vertebrate LSU RPs have much higher sequestration of basic residues than those of bacteria, archaea and even of the lower eukarya. The basic clusters are highly aligned in the vertebrate, but less in the lower eukarya, and only within families in archaea and bacteria. Increase in the basicity of RPs, besides helping transport to the nucleus, should promote stability of the assembled ribosome as well as the association with translocons and other intracellular matrix proteins. The size and GC nucleotide bias of the expansion segments of large LSU rRNAs also culminate in the vertebrate, and should support ribosome association with the endoplasmic reticulum and other intracellular networks. However, the expansion and nucleotide bias of eukaryote LSU rRNAs do not clearly correlate with changes in ionic parameters of LSU ribosomal proteins.

Dimers of G-protein coupled receptors as versatile storage and response units.

The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αßγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.

On the segregation of protein ionic residues by charge type.

Based on ubiquitous presence of large ionic motifs and clusters in proteins involved in gene transcription and protein synthesis, we analyzed the distribution of ionizable sidechains in a broad selection of proteins with regulatory, metabolic, structural and adhesive functions, in agonist, antagonist, toxin and antimicrobial peptides, and in self-excising inteins and intron-derived proteins and sequence constructs. All tested groups, regardless of taxa or sequence size, show considerable segregation of ionizable sidechains into same type charge (homoionic) tracts. These segments in most cases exceed half of the sequence length and comprise more than two-thirds of all ionizable sidechains. This distribution of ionic residues apparently reflects a fundamental advantage of sorted electrostatic contacts in association of sequence elements within and between polypeptides, as well as in interaction with polynucleotides. While large ionic densities are encountered in highly interactive proteins, the average ionic density in most sets does not change appreciably with size of the homoionic segments, which supports the segregation as a modular feature favoring association.

Two intracellular helices of G-protein coupling receptors could generally support oligomerization and coupling with transducers.

For many G-protein coupling receptors (GPCRs), the upkeep of receptor dimers could depend on association with functional Gi α subunits. This is known for Y1, Y2 and Y4 neuropeptide Y receptors [presented in the companion paper (Estes et al., Amino Acids, doi: 10.1007/s00726-010-0642-z , 2010)]. Interactions with transducers use mainly intracellular domains of the receptors. Intracellular loops 1 and 2 in GPCRs are short and lack extensive helicity that could support transducer anchoring. Interaction with G-proteins is known to use the juxtamembrane Helix 8 in the fourth intracellular domain, for which we document a helix-stabilizing n/(n + 4) pattern of large hydrophobic sidechains. Another intracellular helix located in the C-terminal portion of the third intracellular loop does not display a strong stabilizing pattern, and is found in many studies to serve dynamically in association and activation of transducers and effectors. We show that these tracts share features across metazoan phyla not only in opsins and opsin-like receptors (including the Y receptors), but also in Taste-2 and Frizzled receptors. Similarities of these helices across GPCR groups could have both phylogenetic and functional roots.

Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers.

Treatment of CHO cells expressing human Y receptors (Y(1), Y(2) or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).

The fourth intracellular domain of G-protein coupling receptors: helicity, basicity and similarity to opsins.

The minimal size of the fourth intracellular domain of heptahelical G-protein coupling receptors (GPCRs) is close to 15 residues, and a juxtamembrane 15-residue segment is predicted as helical (Helix-8) in most of the receptors. Sequences of opsins, non-visual opsin-like (family A) GPCRs and Taste-2 receptors correspond with bovine rhodopsin at four positions in this tract. This is especially evident in monoamine receptors. In most GPCRs, the conserved juxtamembrane segment also has a large fraction of basic sidechains, and a considerable excess of cationic over anionic residues. The conservation is not dependent on the preferred G-protein alpha subunit or the overall length of the domain, indicating an additive speciation. In rod opsins and some A-GPCRs this segment has been shown to associate with the bilayer and to interact with G-proteins. The segment could also be involved in precoupling of receptors and transducers. These interactions could be helped by both the structural propensities and the high content of cationic sidechains.

Fragmentation and Matching of Human MicroRNA Sequences in 3'utr.

AIMS: Definition of sense and antisense microRNA matches in 3'utr. BACKGROUND: Matches of mature microRNAs (m-miRs) in human 3'utr could be traced to mutations producing fragments of original m-miR sequences without physical separation. (The m-miR matches in 5'utr and cds should be by far fewer, but could follow similar patterns). OBJECTIVE: To ascertain if the sense and antisense m-miR fragments in 3'utr occur at similar or different levels. METHODS: Frequency of sense and antisense m-miR matches in 3'utr was examined in the range of 7-22 nucleotides. RESULTS: The fragmentation occurs at gene level by mutation within one of the paired m-miRs, which upon transcription results in increased interactive capability for both former pre-micro (premir) RNA stem partners. The non-mutated stem partner can persist in 3'utr sequences, as is apparent from significant presence of miR-619-5p and miR-5096 and some conservation of 20 other simian- specific m-miR sequences. However, most of m-mir sequences in 3'utr are extensively fragmented, with low preservation of long matches. In flanks of individual m-miR embeds the mutated pre-mir positions are to a degree defined specifically. CONCLUSION: The m-mir matches of various sizes in 3'utr apparently reflect accumulation, on a phylogenetic time scale, of in-sequence point mutations. Across human 3'utr this fragmentation is significantly less for evolutionarily recent human m-miRs that originate in simians compared to human m-miRs first appearing in lower primates, and especially to human m-miRs introduced in nonprimates.

Pertussis toxin induces parallel loss of neuropeptide Y Y1 receptor dimers and Gi alpha subunit function in CHO cells.

Treatment with pertussis toxin in addition to a stable inhibition of G(i)alpha subunits of G-proteins also strongly reduced human neuropeptide Y Y(1) receptors expressed in Chinese hamster ovary (CHO) cells. This was reflected in abolition of the inhibition by Y(1) agonists of forskolin-stimulated adenylyl cyclase in intact cells, and of Y(1) agonist stimulation of GTPgammaS binding to particulates from disrupted cells. The loss of both receptor and G(i)alpha subunit function was attenuated by ammonium chloride, an inhibitor of acid proteinases, pointing to a chaperoning co-protection of active pertussis toxin-sensitive Galpha subunits and Y(1) receptors. The surface complement of the Y(1) receptor was changed a little in conditions of approximately 85% decrease of the Y(1) population, but the rate of the Y(1) receptor-linked internalization of agonist peptides was reduced about 70%. The preserved receptor fraction consisted of monomers significantly coupled to G(q)alpha subunits. The persistent pertussis toxin-insensitive internalization of agonists with the Y(1) receptor may reflect a rescue or alternative switching that could be important for cell functioning in neuropeptide Y-rich environments. The results are compatible with a loss, due to G(i)alpha subunit inactivation by the toxin, of a large Y(1) receptor reserve constituted of oligomers associating with heterotrimeric G-proteins.

Importance of a N-terminal aspartate in the internalization of the neuropeptide Y Y2 receptor.

With human neuropeptide Y Y2 receptor expressed in the Chinese hamster ovary (CHO) cells, the Asp35Ala mutation, and especially the change of Pro34Asp35 to Ala34Ala35, decrease the compartmentalization and strongly accelerate internalization of the receptor. These changes are not associated with alterations in agonist affinity, G-protein interaction, dimerization, or level of expression of the mutated receptors relative to the wildtype receptor. The proline-flanked aspartate in the N-terminal extracellular segment of the neuropeptide Y Y2 receptor thus apparently has a large role in anchoring and compartmentalization of the receptor. However, the Pro34Ala mutation does not significantly affect the embedding and cycling of the receptor.

The Expansion Segments of Human 28S rRNA Match MicroRNAs Much Above 18S rRNA or Core Segments.

BACKGROUND: The size of eukaryotic 25-28S rRNAs shows a progressive phylogenetically linked increase which is pronounced in mammals, and especially in hominids. The increase is confined to specific expansion segments, inserted at points that are highly conserved from yeast to man. These segments also show a progressive increase in nucleotide bias, mostly the GC bias. Substantial parts of the large expansion segments 7, 15 and 27 of 28S rRNA are known to be exposed at the ribosome surface, with no clear association with ribosomal proteins. These segments could bind extraneous RNAs and proteins to support regulatory events. METHODS: This study examined the possible canonical matching of human 28S rRNA and 18S rRNA segments with 2586 human microRNAs. This was compared with matching of the microRNAs to sectors of 18810 human mRNAs. RESULTS: The overall matching was rather similar across 18S rRNA segments and core segments of 28S rRNA. However, the expansion segments of 28S rRNA (abbreviated ESL) collectively have a much higher (up to two-fold) capacity for the canonical association with microRNAs. This is pronounced in large ESL, and is found to strongly relate to the GC content of microRNAs. CONCLUSION: Oligonucleotides and microRNAs of high GC content through a strong canonical hydrogen bonding could have large activity in regulation of subcellular RNAs. In view of the considerable abundance of ribosomal RNAs in many mammalian tissues, ESL could constitute an important component of microRNA balance, possibly serving to lower the availability of GC-rich microRNAs (and thereby help conservation of GC-rich mRNAs).

The Expansion Segments of 28S Ribosomal RNA Extensively Match Human Messenger RNAs.

Eukaryote ribosomal RNAs (rRNAs) have expanded in the course of phylogeny by addition of nucleotides in specific insertion areas, the expansion segments. These number about 40 in the larger (25-28S) rRNA (up to 2,400 nucleotides), and about 12 in the smaller (18S) rRNA (<700 nucleotides). Expansion of the larger rRNA shows a clear phylogenetic increase, with a dramatic rise in mammals and especially in hominids. Substantial portions of expansion segments in this RNA are not bound to ribosomal proteins, and may engage extraneous interactants, including messenger RNAs (mRNAs). Studies on the ribosome-mRNA interaction have focused on proteins of the smaller ribosomal subunit, with some examination of 18S rRNA. However, the expansion segments of human 28S rRNA show much higher density and numbers of mRNA matches than those of 18S rRNA, and also a higher density and match numbers than its own core parts. We have studied that with frequent and potentially stable matches containing 7-15 nucleotides. The expansion segments of 28S rRNA average more than 50 matches per mRNA even assuming only 5% of their sequence as available for such interaction. Large expansion segments 7, 15, and 27 of 28S rRNA also have copious long (≥10-nucleotide) matches to most human mRNAs, with frequencies much higher than in other 28S rRNA parts. Expansion segments 7 and 27 and especially segment 15 of 28S rRNA show large size increase in mammals compared to other metazoans, which could reflect a gain of function related to interaction with non-ribosomal partners. The 28S rRNA expansion segment 15 shows very high increments in size, guanosine, and cytidine nucleotide content and mRNA matching in mammals, and especially in hominids. With these segments (but not with other 28S rRNA or any 18S rRNA expansion segments) the density and number of matches are much higher in 5'-terminal than in 3'-terminal untranslated mRNA regions, which may relate to mRNA mobilization via 5' termini. Matches in the expansion segments 7, 15, and 27 of human 28S rRNA appear as candidates for general interaction with mRNAs, especially those associated with intracellular matrices such as the endoplasmic reticulum.

Canonical microRNA Matching Differs Greatly Across Groups of G-protein Coupled Receptor mRNAs.

BACKGROUND: Heptahelical G protein coupled receptors (GPCRs) support numerous sensory and metabolic functions and differ considerably in levels of expression. GPCR protein levels should link to regulation of GPCR mRNAs by microRNAs (miRs), which might significantly depend on numbers, size and GC content of the canonical antisense matches in mRNAs. These parameters of GPCR mRNAs have not been studied in detail. METHODS: Canonical matching profiles of human GPCR mRNAs and miRs were examined using segments of 7-15 nucleotides in windows shifted by one position over the entire microRNA sequence. RESULTS: Human GPCRs mRNAs within larger function-related groups have a quite homogenous matching with miRs. Both the GC content and the melting temperature (and hence also the binding energy) are appreciably higher in 5'utr compared to 3'utr matches of the same length. Increase in the GC content correlates significantly with length in the ubiquitous matches of 7-12 nucleotides. However, several GPCR groups strongly differ in overall match numbers and density. The untranslated regions of sensory receptor mRNAs, especially the olfactory and Taste-2 mRNAs, have the lowest match numbers and density and the fewest miR partners. The glucagon and frizzled families show the highest canonical matching. CONCLUSION: Partnership of GPCR mRNAs and miRs could significantly relate to the type of function of the receptor proteins, with mRNAs of the sensory receptors having the lowest and those of metabotropic GPCRs the highest targeting. This could be of interest regarding GPCR regulation by exogenous miRs.

Canonical Matches of Human MicroRNAs with mRNAs: A Broad Matrix of Position and Size.

BACKGROUND: Canonical hydrogen-bonding multi-nucleotide matches of microRNAs (miRs) with mRNAs are considered as important in mRNA regulation. MiR "seed" positions 2-8 are frequently viewed as mRNA partners, but there is ample evidence for use of other (and even non-contiguous) miR parts. No detailed information is available about canonical matching, and the GC content of the matches is rarely considered, although it should have a major regulatory potential. METHODS: Sequences of 2586 human miRs and of 5'utr, cds and 3'utr in 18810 human mRNAs were examined for number and GC content of contiguous Watson-Crick antisense matches of six or more nucleotides (nt) in successive windows shifted by 1 nt. RESULTS: Frequency of the antisense matches is within all sectors similar for segments of up to 10 nt starting at positions 1-10 of miR sequences, with decrease of 3.5 to 4-fold for each 1-nt increment. Adenine and uracil rich elements (ARE-like) are very frequent in cds and 3'utr. All mRNAs have matches of up to 10 nt, and most also those of 11-15 nt. The match density is largest in 5'utr, and the match number in cds. The 5'utr and cds matches average much higher GC content than those of 3'utr. The GC content of matches is above that for the whole sector in 5'utr and cds, but lower in 3'utr. CONCLUSION: Human mRNA matches across miR sequences constitute a positionally similar matrix of canonical hydrogen-bonding reactivity. This presents ample opportunities for contiguous binding independent of miR position. The ubiquitous 10 to 15-nt matches could serve as binding foci. Interaction of miRs with the abundant GC-rich 5'utr and cds counterparts could be important in the regulation of mRNA-ribosome interaction as well as in mRNA disposal. The lower density and GC content of a majority of 3'utr matches could mainly support a dynamic regulation by miRs.

Neuropeptide Y as a partial agonist of the Y1 receptor.

In absence of receptor cycling, human/rat neuropeptide Y was found to persistently occupy the guinea pig neuropeptide Y Y1 receptors expressed on the surface of Chinese hamster ovary (CHO) cells (IC50 approximately 8 nM); a lasting occupancy was also evident with active receptor cycling. A similar blockade was obtained with the human neuropeptide Y Y1 receptor (in CHO or SK-N-MC cells). Peptidic antagonists GR238118 (1229U91) and VD-11 blocked the Y1 receptor in the same molarity range. A neuropeptide Y-related Y1 agonist, (Leu31Pro34) human neuropeptide Y, also strongly adhered to the Y1 site. Similar blockade-like occupancy by neuropeptide Y was found with particulates from Y1-expressing CHO cells, and with native neuropeptide Y Y1 receptors of rat synaptosomes. Peptide YY and a related Y1-selective agonist, (Leu31Pro34) human peptide YY, showed a much less stable binding to the neuropeptide Y Y1 receptor with either the intact cells or particulates. The Y1 binding of neuropeptide Y was also less sensitive to chaotropic agents and guanine nucleotides than the binding of peptide YY, indicating a larger stability for association of neuropeptide Y with the receptor. Inhibition of forskolin-stimulated adenylyl cyclase showed a distinctly attenuating agonism for neuropeptide Y, with an activity similar to peptide YY below 1 nM, but considerably lower above 3 nM of the peptides. This activity was largely exerted via pertussis toxin-sensitive G-proteins of Y1-CHO cells. Our findings indicate that signaling by neuropeptide Y via its Y1 receptor could be self-restricting at higher levels of the peptide, in relation to a strong association of the agonist with the Y1 binding site.

Lithium inhibits internalization and endosomal processing of both neuropeptide Y (NPY) Y1 and transferrin receptors.

Low concentrations of Li+ reduce the rate of internalization of neuropeptide Y (NPY) Y1 receptors [M.S. Parker, S.L. Parker, J.K. Kane, Internalization of neuropeptide Y Y1 and Y5 and of pancreatic polypeptide Y4 receptors is inhibited by lithium in preference to sodium and potassium ions, Regul. Pept., 118 (2004) 67-74]. This Li+-induced decrease in Y1 receptor internalization could be alleviated by Y1 receptor agonists. As shown by fractionation on Percoll gradients, lithium treatment induces a concentration-related decrease of intermediate and higher endosomal densities that contain the internalized Y1 ligand-receptor complex. This indicates an inhibition of endosome processing and maturation. Internalization of human transferrin shows [Li+] sensitivity similar to that of the Y1 receptor, and a similar Li+-induced decrease in endosomal processing. Lithium treatment thus decreases activity of the endosome system shared in the recycling endocytosis of the Y1 and transferrin receptors.

G and C Iterons and Strings in MicroRNAs Should be Important in Regulation of mRNAs(†).

BACKGROUND: Same-nucleotide repeats (iterons) are strongly expressed in many DNA regions and RNA classes. These repeats serve importantly in association of polynucleotides and proteins, but have not been characterized in miRNAs. METHODS: Iterons and nucleotide strings were quantified in currently known human miRNAs, including some comparisons with miRNAs of other species. RESULTS: Human 5p miRNAs have significantly more G iterons than other miRNA groups. The 3p miRNAs have an inverse excess of C iterons. The miRNAs lacking functional counter-stems (which we differentiate as 5n or 3n by position in pre-miRNAs) also have a large excess of G iterons. In 5p miRNAs G and C iterons have much higher density in the seed compared to the post-seed region. This difference is lower in 5n and 3n sequences, and much lower in 3p sequences. In all groups the contiguous GC strings constitute a larger part of sequences than the AU strings. A surplus of G or C iterons and of GC strings should enable a more stable association with the target mRNAs. CONCLUSION: From the available evidence, the G iteron- and GC-rich miRNAs should also interact more readily with miRNA-processing and similar proteins.

Homoiterons and expansion in ribosomal RNAs.

Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks.

Pancreatic polypeptide receptors: affinity, sodium sensitivity and stability of agonist binding.

Cloned rat, human and guinea-pig Y4 pancreatic polypeptide (PP) receptors expressed in Chinese hamster ovary (CHO) cells, as well as the rabbit Y4-like PP receptor, show a selective sensitivity to Na+ over K+ ion in PP attachment, but little sensitivity to Na+ in dissociation of bound PP peptides. Agonist binding to Y4 receptors of intact CHO cells also shows much greater sensitivity to Na+ over K+, and a tenacious attachment of the bound agonist. Binding sensitivity to K+ is greatly enhanced upon receptor solubilization. Pancreatic polypeptide sites also show large sensitivity to modulators of Na+ transport such as N5-substituted amilorides and to RFamides, as different from Y1 or Y2 receptors. Thus, PP binding is modulated by cation-induced changes in site environment (with selectivity for Na+) and ultimately results in a blocking attachment. This would support receptor operation in the presence of ion gradients, as well as prolonged agonist-delimited signaling activity (which can include partial antagonism). Also, this could point to an evolutionary adaptation enabling small numbers of PP receptors to perform extensive metabolic tasks in response to low agonist signals.

Internalization of neuropeptide Y Y1 and Y5 and of pancreatic polypeptide Y4 receptors is inhibited by lithium in preference to sodium and potassium ions.

The receptor-linked internalization of [125I] human neuropeptide Y (NPY) in Chinese hamster ovary (CHO) cells expressing the guinea-pig Y1 receptors or in human endometrial carcinoma-1B (Hec-1B) cells expressing the human Y5 receptor, as well as the receptor-linked internalization of human pancreatic polypeptide (hPP) receptor expressed in CHO cells, is selectively inhibited by low molarities of the Li+ cation. The Na+ and K+ cations decreased the receptor-linked internalization of agonist peptides only at high molar inputs, and largely in proportion to the reduction of cell surface binding of Y ligand peptides, dependent on ion concentration and the type of Y receptor examined. With particulates isolated from disrupted cells, there was no preferential inhibition by Li+ relative to Na+ in the binding of type-specific ligand peptides to Y receptors of any type. The observed difference could be connected to the known ability of Li+ to modify active conformations of signal transducers, which may also directly or indirectly affect the internalization motors. The decrease in the rate of Y receptor internalization by Li+ also points to a possible alteration of Y receptor signaling in vivo by lithium at acute therapeutically employed dose levels.

Internalization of pancreatic polypeptide Y4 receptors: correlation of receptor intake and affinity.

Unlike neuropeptide Y receptors, the pancreatic polypeptide Y4 receptors display considerable differences in sequence and ligand-binding affinity across mammalian species. This could produce different receptor turnover rates in the same cellular membrane environment. Comparing rat, human and guinea-pig Y4 receptors expressed in Chinese hamster ovary (CHO) cells (K(d) with human pancreatic polypeptide 14, 45 and 116 pM, respectively), we indeed found human pancreatic polypeptide internalization in the rank order of receptor affinities. A large fraction of the internalized human pancreatic polypeptide, similar across the Y4 species, was associated with secondary endosomes (density approximately 1.05 in Percoll gradients) and lysosomes (density approximately 1.11). For all Y4 receptors examined, this intake was potently and selectively inhibited by cholesterol-complexing polyene antibiotic filipin III and also by clathrin lattice formation inhibitor, phenylarsine oxide. Internalization differences found across Y4 receptor species to a degree compare with those observed for the cloned guinea-pig neuropeptide Y Y1 and human neuropeptide Y Y5 receptors and, generally, support ligand-binding affinities as important determinants of internalization for neuropeptide receptors.