Identification of structural requirements for analogs of atrial natriuretic peptide (ANP) to discriminate between ANP receptor subtypes

Abstract

The structure-activity relationships for affinity and selective binding of atrial natriuretic peptide (ANP) and analogues to guanylate cyclase coupled (CC) and non-cyclase coupled (NC) receptors in rabbit lung membranes are described. We have designed a series of peptides to try to identify the minimal sequence involved in specific recognition of each receptor subtype. The affinity of the peptides was determined from competitive binding experiments. Several peptides derived from the rat ANP sequence, e.g., des-[Phe106, Gly107, Ala115, Gln116]ANP-(103-125)NH2 (4), des-[Cys105,121]ANP-(104-126) (5), and [Acm-Cys105]ANP-(105-114)NH2 (9) have high affinity and selectivity for the noncoupled site. Peptide 4 was the most selective ligand with an affinity superior to that of ANP-(103-126). This compound does not displace the radiolabeled ligand from the guanylate cyclase coupled receptor at the highest concentration tested (100 nM). The structure-activity relationship for affinity and selectivity is discussed. Comparison of the peptide sequences suggests that the structural feature responsible for recognition of the NC site resides in a single sequence of seven contiguous amino acids from the cyclic core of the hormone. The corresponding heptapeptide retains affinity to the guanylate cyclase uncoupled binding site and is proposed to encompass the minimal sequence for specific recognition of the non-guanylate cyclase coupled ANP receptor.

Atrial natriuretic factor receptor subtypes in the rat central nervous system.

“…In addition, we used another ligand selective for the ANF-C receptor, namely, ANF-(106-113)-NH 2 , whose amino acid sequence encompasses the structural feature responsible for recognition of the ANF-C receptor. 6 This peptide retains a high affinity (K b 2-15 nM) for the ANF-C receptors in cultured vascular smooth muscle cells and isolated perfused kidney 26 and inhibits adenylate cyclase activity and cyclic AMP levels in vascular smooth muscle cells from rat aorta with a potency similar to that of C-ANF and ANF. 9 Analysis of the competition curves revealed, however, that C-ANF inhibition of 125 I-ANF binding in CNS structures, with the exception of the PA and ChP, had the characteristics of interaction with the ANF-A or ANF-B rather than the ANF-C receptor (ICJO>1 /iM).…”

“…These two receptor subtypes impose stringent constraints on ligand structure for binding and stimulation of cyclic GMP. 6 The ANF-C receptor subtype is a non-guanylate cyclase-containing receptor, 5 existing on the cell membrane predominantly as a homodimer of 60-70 kDa subunits. 1^7 This receptor subtype binds native ANF as well as a range of truncated, ring-deleted and linear ANF analogues with high affinity.…”

“…1^7 This receptor subtype binds native ANF as well as a range of truncated, ring-deleted and linear ANF analogues with high affinity. 6 One of the first synthetic ligands showing selectivity for the ANF-C receptor was C-ANF (des-[Gln ]-NH 2 ). 8 More recently, a number of linear peptides corresponding to various fragments of the ANF sequence have been shown to retain nanomolar affinities for ANF-C but not for ANF-A or ANF-B receptors, thus being able to discriminate between guanylate cyclase-containing and non-guanylate cyclase-containing ANF receptor subtypes.…”

“…8 More recently, a number of linear peptides corresponding to various fragments of the ANF sequence have been shown to retain nanomolar affinities for ANF-C but not for ANF-A or ANF-B receptors, thus being able to discriminate between guanylate cyclase-containing and non-guanylate cyclase-containing ANF receptor subtypes. 6 In fact, linear peptides as short as seven to 10 amino acids in length, encompassing residues 109-113 of rat ANF, are endowed with selective ANF-C receptor binding abilities.…”

“…The cryostat tissue sections then were preincubated at room temperature for 10 minutes in 50 mM Tris-HCl buffer (pH 7.5) containing 0.5% bovine serum albumin and were incubated for 90 minutes in the same buffer supplemented with 100 pM 125 I-ANF and the appropriate concentrations of unlabeled ligands (see below), as well as 10" 6 I-ANF binding, a set of sections was incubated in the presence of 10~6 M angiotensin II, glucagon, or substance P. For competition analysis, the incubation buffer contained either unlabeled ANF, C-ANF, or ANF-(106-113)-NH 2 in concentrations ranging from 10~1 2 to 10~5 M. After incubation, the slides were washed (two times, 10 minutes each, 4°C) in 50 mM Tris-HCl (pH 7.5) containing 0.5% bovine serum albumin, were fixed (15 minutes) in 2% glutaraldehyde (pH 7.5, 4°C), were washed (5 minutes) in 0.1 M phosphate buffer (pH 7.5, 4°C) and in distilled water, and were dehydrated in alcohol and dried overnight at 60°C. Subsequently, the sections along with I standards were apposed to x-ray films for 2-3 days at room temperature.…”