A Human Orphan Calcitonin Receptor-like Structure

117

To define the structural requirements of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor necessary for activation of phospholipase C (PLC), receptors with random mutations in their second cytoplasmic loop were synthesized, and their properties were assessed. A mutant in which the wild type (WT) rat PTH/PTHrP receptor sequence EKKY (amino acids 317-320) was replaced with DSEL had little or no PTH-stimulated PLC activity when expressed transiently in COS-7 cells, but it retained full capacity to bind ligand and to generate cAMP. This phenotype was confirmed in LLC-PK1 cells stably expressing the DSEL mutant receptor, where both PTH-stimulated PLC activity and sodium-dependent phosphate co-transport were essentially abolished. Individual mutations of these four residues point to a critical role for Lys-319 in receptor-G protein coupling. PTH-generated IPs were reduced to 27 ؎ 13% when K319E, compared with the WT receptor, and PLC activation was fully recovered in a receptor revertant in which Glu-319 in the DSEL mutant cassette was restored to the WT residue, Lys. Moreover, the WT receptor and a mutant receptor in which K319R had indistinguishable properties, thus suggesting that a basic amino acid at this position may be important for PLC activation. All of these receptors had unimpaired capacity to bind ligand and to generate cAMP. To ensure adequacy of G␣ q -subunits for transducing the receptor signal, G␣ q was expressed in HEK293 and in LLC-PK1 cells together with either WT receptors or receptors with the DSEL mutant cassette. PTH generated no inositol phosphates (IPs) in either HEK293 or LLC-PK1 cells, when they expressed DSEL mutant receptors together with G␣ q . In contrast, PTH generated 2-and 2.5-fold increases in IPs, respectively, when these cells co-expressed both the WT receptor and G␣ q . Thus, generation of IPs by the activated PTH/PTHrP receptor can be selectively abolished without affecting its capacity to generate cAMP, and Lys-319 in the second intracellular loop is critical for activating the PLC pathway. Moreover, ␣-subunits of the G q family, rather than ␤␥-subunits, transduce the signal from the activated receptor to PLC, and the PLC, rather than the adenylyl cyclase, pathway mediates sodium-dependent phosphate cotransport in LLC-PK1 cells.Signals from the parathyroid hormone (PTH) 1 /PTH-related peptide (PTHrP) receptor are transduced by G proteins. The lack of sequence homology between PTH/PTHrP receptors and all but a few of the other G protein-coupled receptors, however, justifies classifying them as members of a distinct family (1), which includes two mammalian receptors each for PTH or PTHrP (2, 3), vasoactive intestinal peptides (4, 5), and corticotrophin-releasing hormone (6), and receptors for secretin (7), calcitonin (8), glucagon-like peptide 1 (9), growth hormonereleasing hormone (10), glucagon (11), pituitary adenylyl cyclase-activating peptide (12), gastric inhibitory peptide (13), and an orphan receptor in brain similar to the calcitonin receptor (14). Recep...

Section: Discussionsupporting

confidence: 89%

mentioning

confidence: 99%

Mutations in the Second Cytoplasmic Loop of the Rat Parathyroid Hormone (PTH)/PTH-related Protein Receptor Result in Selective Loss of PTH-stimulated Phospholipase C Activity

Iida-Klein

Guo

Takemura

et al. 1997

117

“…The levels of hCRLR, hRAMP, and hAM mRNA were normalized to those of glyceraldehyde-3-phosphate dehydrogenase mRNA, which served as an internal control. DNA sequence analysis confirmed that the amplified products were identical to those of hCRLR and the three hRAMP isoforms (6,19,20).…”

Section: Methodsmentioning

confidence: 71%

Identification of the Human Receptor Activity-modifying Protein 1 Domains Responsible for Agonist Binding Specificity

Kuwasako

Kïtamura

Nagoshi

et al. 2003

When co-expressed with receptor activity-modifying protein (RAMP) 1, calcitonin receptor-like receptor (CRLR) can function as a receptor for both calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). To investigate the structural determinants of ligand binding specificity, we examined the extracellular domain of human (h) RAMP1 using various deletion mutants. Co-expression of the hRAMP1 mutants with hCRLR in HEK-293 cells revealed that deletion of residues 91-94, 96 -100, or 101-103 blocked [ 125 I]CGRP binding and completely abolished intracellular cAMP accumulation normally elicited by CGRP or AM. On the other hand, the deletion of residues 78 -80 or 88 -90 significantly attenuated only AM-evoked responses. In all of these cases, the receptor heterodimers were fully expressed at the cell surface. Substituting alanine for residues 91-103 one at a time had little effect on CGRPinduced responses, indicating that although this segment is essential for high affinity agonist binding to the receptors, none of the residues directly interacts with either CGRP or AM. This finding suggests that RAMPs probably determine ligand specificity by contributing to the structure of the ligand-binding pocket or by allosteric modulation of the conformation of the receptor. Interestingly, the L94A mutant up-regulated surface expression of the receptor heterodimer to a greater degree than wild-type hRAMP1, thereby increasing CGRP binding and signaling. L94A also significantly increased cell surface expression of the hRAMP1 deletion mutant D101-103 when co-transfected with hCRLR, and expression of a L94A/D101-103 double mutant markedly attenuated the activity of endogenous RAMP1 in HEK-293T cells.CGRP 1 and AM belong to the calcitonin family of regulatory peptides and are both highly potent vasodilators (1, 2). Although they share very little sequence identity, both contain ring structures comprised of six amino acids linked by a disulfide bridge and an amidated C terminus that are required for biological activity (3). Both of these peptides and their specific or common receptors are widely distributed among peripheral tissues and in the central nervous system, enabling them to exert a wide variety of biological effects (4, 5).RAMPs are a recently identified group of single transmembrane domain accessory proteins that serve to transport CRLR to the cell surface where they form functional CGRP and AM receptors (6). The three RAMP isoforms (RAMP1, RAMP2, and RAMP3), which share only 30% sequence identity and differ in their tissue distributions, are all comprised of ϳ160 amino acids that make up a large extracellular N-terminal domain, a single membrane-spanning domain, and a very short cytoplasmic domain (6, 7). Co-expression of CRLR with RAMP1 leads to both proteins being presented at the plasma membrane as a heterodimeric CGRP receptor, whereas co-expression of CRLR with RAMP2 or RAMP3 enables the resultant heterodimer to function as a AM receptor (6,8,9). Upon binding their respective agonist, both receptors mediate a rise...

“…We now know that the CGRP receptor is unique among G-protein-coupled receptors in that it is a complex composed of at least three proteins. The ligand-binding protein named calcitonin receptor-like receptor (CRLR) has the stereotype structure of a seven-transmembrane receptor but is inactive when expressed in cells alone (29,51). CRLR requires two additional accessory proteins for function; receptor activity modifying protein 1 (RAMP1) acts as a molecular chaperone and is required for routing of CRLR to the cell surface as well as pharmacologic specificity (27), and the CGRP receptor component protein (RCP) is required for coupling the receptor to the cellular signal transduction pathway (28,52).…”

mentioning

confidence: 99%

Localization of the Calcitonin Gene-related Peptide Receptor Complex at the Vertebrate Neuromuscular Junction and Its Role in Regulating Acetylcholinesterase Expression

Rossi¹,

Dickerson

Rotundo

2003

The calcitonin gene-related peptide (CGRP) is released by motor neurons where it exerts both short and long term effects on skeletal muscle fibers. In addition, sensory neurons release CGRP on the surrounding vasculature where it is in part responsible for local vasodilation following muscle contraction. Although CGRPbinding sites have been demonstrated in whole muscle tissue, the type of CGRP receptor and its associated proteins or its cellular localization within the tissue have not been described. Here we show that the CGRPbinding protein referred to as the calcitonin receptorlike receptor is highly concentrated at the avian neuromuscular junction together with its two accessory proteins, receptor activity modifying protein 1 and CGRP-receptor component protein, required for ligand specificity and signal transduction. Using tissue-cultured skeletal muscle we show that CGRP stimulates an increase in intracellular cAMP that in turn initiates down-regulation of acetylcholinesterase expression at the transcriptional level, and, more specifically, inhibits expression of the synaptically localized collagen-tailed form of the enzyme. Together, these studies suggest a specific role for CGRP released by spinal cord motoneurons in modulating synaptic transmission at the neuromuscular junction by locally inhibiting the expression of acetylcholinesterase, the enzyme responsible for terminating acetylcholine neurotransmission.The calcitonin gene-related peptide (CGRP) 1 is a 37-amino acid neuropeptide expressed in a wide variety of neurons in the central and peripheral nervous systems, including motoneurons (reviewed in Refs. 1-4), where it is released upon stimulation (5). In skeletal muscle, CGRP has been shown to potentiate muscle contraction (6), increase the rate of acetylcholine receptor (AChR) desensitization (7-10), increase the numbers of AChR (11), decrease the levels of acetylcholinesterase (AChE) (12)(13)(14), and locally increase the rate of blood flow following muscle contraction (15)(16)(17). Although the presence of CGRP in nerve terminals at the neuromuscular junction has been well documented (18 -21), the localization of CGRP receptors at the synapse has been more difficult to demonstrate because the only available probe was the receptor ligand itself, CGRP. Using 125 I-CGRP as a probe, high affinity CGRP receptors were shown to be expressed on the surfaces of tissuecultured myotubes (22) and present on whole muscle membranes isolated from embryonic or newly hatched chicks (23); however, the latter studies could not differentiate between CGRP receptors on skeletal muscle versus those on the surrounding vasculature. In one autoradiographic study (24), a correlation between 125 I-CGRP and AChE histochemical staining was observed in low power photomicrographs of rat bulbocavernosus muscle, suggesting an accumulation of CGRP receptors at sites of nerve-muscle contact. However, in these studies it was not possible to differentiate between CGRP receptors located in blood vessels or Schwann cells and those on m...