Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR.
G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.
Conner
Matthew
M
School of Biosciences, University of Birmingham, UK.
Hicks
Matthew R
MR
Dafforn
Tim
T
Knowles
Timothy J
TJ
Ludwig
Christian
C
Staddon
Susan
S
Overduin
Michael
M
Günther
Ulrich L
UL
Thome
Johannes
J
Wheatley
Mark
M
Poyner
David R
DR
Conner
Alex C
AC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D015740:Calcitonin Gene-Related Peptide / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D018015:Receptors, Calcitonin Gene-Related Peptide; D015740:Calcitonin Gene-Related Peptide