New evidence for a membrane-bound pathway in hormone receptor binding

Abstract

The fully active cholecystokinin analog (Thr,Nle)-CCK-9 was lipo-derivatized by N-terminal grafting of a dimyristoylglycerol moiety to induce tight interdigitation with cell membrane bilayers. While the parent CCK peptide was shown to interact only transiently with small unilamellar phospholipid vesicles, the lipo-CCK peptide, although self-aggregating into vesicles, inserts rapidly and quantitatively into phospholipid bilayers. Fluorescence and, even more so, NMR data are supportive for a chain reversal of the CCK moiety of the lipo derivative with embedment of the C-terminus into hydrophobic compartments of the bilayer. MD simulations allowed for a proposal of the folded form of CCK in bilayers with a helical array parallel to the interface and an amphipathic display of the side chains. In this model, the phenylalanine aromatic ring is heading the peptide molecule and may thus play a decisive role in the lateral penetration of the receptor at the water/lipid interface. In fact, despite the membrane-bound state, its binding affinity for rat pancreatic acini is comparable to that of the CCK peptide when tested after a 3-h equilibration period but 5-6-fold lower at 45 min, suggesting that the association rate is significantly lower than that of the unmodified CCK peptide. This can rationally be attributed to the tight interdigitation of the double-tailed lipo moiety with the membrane bilayer. Moreover, an escape of the lipopeptide into the extracellular aqueous phase is energetically highly unfavored; therefore, the receptor can only be reached by a membrane-bound two-dimensional migration. The observed difference in amplification between binding and amylase secretion may result from inadequate occupation of low-affinity CCK receptors, which leads then to poor couplings to G-proteins. Nevertheless the data confirm that lateral penetration of receptor structures is possible, and thus, preadsorption of peptide (neuro)hormones at the cell membrane bilayer may indeed represent the first step in the receptor recognition process.

Expression, Purification, and Characterization of a Soluble Form of the First Extracellular Domain of the Human Type 1 Corticotropin Releasing Factor Receptor

“…In both cases, the K i values of astressin and urocortin for the bNT- CRFR are ϳ5-10-fold lower than those for the wild type receptor and also than those for the CRFR/activin-R chimera (21). The reduced affinity of astressin and urocortin for bNT-CRFR1 may be due to the absence of ligand interactions with the plasma membrane (46). In addition, it is possible that a higher entropic price is required for binding of two soluble proteins compared with that for binding a protein anchored in the membrane.…”

Addressing the Tertiary Structure of Human Parathyroid Hormone-(1–34)

“…If the two helices, and the consequent amphipathic exposure of the amino acids, are relevant elements in the bioactive conformation, the interaction with the cellular membrane could present to the receptor a ligand folded into a preferred conformation (25,58). Similar to the results obtained in water and saline solution the two helical domains are separated by a region of flexibility, in this case centered about residues 18 and 19 (i.e.…”

“…brane-ligand interaction and membrane-induced conformation have been proposed as an operational pathway for the interaction between peptide hormones and their receptors (25)(26)(27)(28).…”

NMR studies of the structure and dynamics of peptide E, an endogenous opioid peptide that binds with high affinity to multiple opioid receptor subtypes

“…Recently, additional support for a membrane-mediated receptor agonist action elicited by peptide hormones has been provided by studies of peptide hormones conjugated to lipids. 8 The membrane anchored peptides were found to have comparable activity to their unmodified counterparts, suggesting a receptor binding site accessible from the membrane.…”

“…A membrane-mediated mechanism of opioid or more generally peptide hormone agonist activity has long been proposed 7 and there is a growing body of evidence to support this hypothesis. 8,50 a Errors in the calculated relaxation rates are determined from the noise level of the spectra and a gaussian fitting analysis of the 2D spectra.…”