Functional data and Strategy

Search Modes

This web application aims to provide tools for investigation of GPHR mutations. Three different general search areas are provided:

• Search Single Mutations
• Search Multiple Mutations
• Search Natural Mutations

All results will be displayed with facilities for further analysis. A detailed description of these facilities can be found here.

Furthermore, in the Search Single Mutations section four different search modes for accessing the mutation data are provided:

• Alignment Search
• Search Single Mutations
• Constitutively Activating Mutations
• 3D Structural Search

Alignment Search

This page contains all GPHRs and their sequences aligned to each other for a quick search and overview of available mutations. Mutations are indicated by a link to the functional data at the appropriate position. For all receptors the secondary structure elements are indicated (e.g. transmembrane helices), as well as the conserved residue of the transmembrane region (see Ballesteros and Weinstein (1995) Methods Neurosci 25, pp. 366-428).

To open the Receptor Alignment Overview webpage click Search Single Mutations⇒ Alignment Search in the menu section.

Search Single Mutations

The Detailed Search mode offers more sophisticated search options:

• Mutation at a specifc position
• Mutations within a restricted region
• Naturally occurring mutations identified and described to be potentially related to pathogenic activation or inactivation
• Mutations substituting amino acids of given properties (e.g. a hydrophilic residue is substituted for a hydrophobic)
• Mutations characterized by a certain functional property

The web application guides through the process of querying. Clicking on Search Single Mutation⇒ Mutation Search, one or more receptors are to select (more than one receptor can be selected by holding the CTRL key while clicking on the receptors).

A second search page helps to specify the query. To find a mutation at a certain position, the option Residue number can be used. The option Sequence region allows finding mutations in a certain region of a receptor. (Any position information have to be given including a potential signal peptide). The first two search options are only available after selecting a single receptor on the first query webpage.

The Functional parameter option can be selected if a search for certain phenotypes or amino acid substitutions is intended. This option allows selecting sub-structure elements such as transmembrane helices.

The last option Naturally occurring finding enables searches for naturally occurring mutations that are described to be related to inactivation or activation of receptors.

If the third option Functional parameter has been choosen, additional information is required. In that case a third webpage appears.

The optional last page allows to specify the amino acid properties of the mutant and wild-type residues

A second option Functional parameter asks to specify phenotypes. For each phenotype a lower and an upper threshold can be selected. These thresholds are numeric percentage values, indicating the mutation phenotype in relation to the wild-type. At least a single functional parameter has to be specified. If specifying more than one functional parameter mutations meeting all requirements will be extracted. Information on the options of the result webpage can be found here.

Constitutively Activating Mutations

Constitutively Activating Mutations are characterized by an increased basal (TSH independent) Gas mediated signaling activity. An alignment (Search Single Mutations⇒ Constitutively Activating Mutations) is available where amino acids characterized by single substitutions with increased basal Gas activity (cAMP signaling activity => 180% compared to wildtype (100%)). This value is chosen by the authors to extract CAMs with significantly increased activity. For more detailed information about the mutation click on the link to get to the Result page

3D Structural Search Search Single Mutations⇒ 3D Structural Alignment This search function allows selection of a receptor and a structural domain (Leucine-rich repeat domain or Serpentine domain). The applet shows a homology model based on the inactivated X-ray structure of bovine rhodopsin. For the modeling process recently published methods were used (Kleinau et al. 2006, JBC). The receptor is shown in backbone representation and all amino acid side-chains are highlighted. Positions where mutation data is available, are indicated by a link at the appropriate position of the wildtype C-atom position of the mutated positions (red balls). By clicking on one or more residues in the applet (click on the red balls, a yellow halo appears around the ball) and on the Identify positions button they will be identified. A list of positions will be shown and Get Results brings up a Result page. This tool gives the possibility to visualize the potential location of amino acids and mutations in the 3D conformation.

In the Search Multiple Mutations section three different search modes for accessing the mutation data are available:

• Overview Double Mutations
• Overview Triple Mutations
• Search Multiple Mutations

Overview Multiple Mutations

For double and triple mutation the same querying process is used. After clicking on Search Multiple Mutations⇒ Overview Double Mutations / Search Multiple Mutations⇒ Overview Triple Mutations in the menu section a receptor can be selected.

At the result page (after Next button) the double or triple mutation is more detailed described by its positions and corresponding substructural location, as well as details (click on details link) about the single mutations, the multiple mutation and experimental conditions. For visualization a 3D model is linked (click on the 3D model link) to each multiple mutation to show the spatial locations in the homology model of the receptor.

Search Multiple Mutations

The Detailed Search mode offers more sophisticated search options. It allows finding:

• Mutation at a specific position
• Mutations within a region

After clicking on Search Multiple Mutations⇒ Search Multiple Mutations in the menu section, a receptor has to be selected, and a second search page asks for one of two different information. To find a double mutation at a certain position, the option Residue number can be used. The option Sequence region allows finding mutations in a certain region of a receptor. At the result page the double or triple mutation is characterized by its positions and corresponding substructural location, as well as details (details link) about the single mutations, the multiple mutation and experimental conditions. For visualization a 3D model is linked (3D model link) to each multiple mutation to show the spatial location in the receptor.

The Natural Mutations menu offers a receptor alignment overview to identify positions of naturally occurring mutations:

Alignment Search

The alignment serves as an overview of available naturally occurring single mutations. The present overview is focused on single side chain substitutions and the first reported publication for each available mutation is linked to PubMed/Medline citation, although there are sometimes more publications for a certain mutation available.Amino acids identified at positions for naturally occurring activating mutations (gain-of-function) are marked green and linked to a more detailed Result page. Naturally occurring inactivating mutations (loss-of-function) are marked red. Positions where both, activating and inactivating mutations are known, are colored magenta.

Result Analysis

The results webpage comprises four sections.

1. a box summarising the inital query
2. right below the text box a table giving an overview over the numbers of mutations that were found
3. a table enabling further analysis (this highlighting table is described in detail below)
4. the last section contains detailed result tables for each receptor.

The receptor information is given at the beginning of each table. It also links to the three-dimensional model of the receptor a 3D morph (both described below), and the according entry in the Swissprot database. The result table itself contains information about the mutation (position, wild-type and mutant residues) and the phenotype. A click on the details element on each line reveals further information about selected mutation.

The details pop-up elaborates on the mutation and specifies information like the celltype used in the studies. For a more detailed reference, it links to the according study in Pubmed. The details pop-up also gives information about mutagenesis experiments performed on corresponding positions of homologue receptors, means on positions that correspond to each other in the multiple sequence alignment held in the database.

Highlighting result values

The highlighting facility allows to specify a lower and upper threshold for each measured functional parameter. Results below the lower threshold can be highlighted in red and results exceeding the upper threshold in green. The tresholds can be individually choosen to extract or visualize certain specific properties.

Projection on three-dimensional receptor model or crystal structure

The projection of the query results on a three-dimensional model is one additional feature. Using the link brings up a new page with a three-dimensional model of the receptor. Since there are only crystal structures available for the LRRD of the hFSHR and the hTSHR, homology models of LRRDs were created for remaining receptors and all receptor serpentine domains ( transmembrane helices and connecting loops). If there are mutations outside these domain, e.g. in the C-terminal tail, they can't be projected on the model.

For all receptor serpentine domains homology models in the activated conformation (based on X-ray structure of bovine opsin) were generated based on the modeling protocoll described in Kleinau et al., 2008, CMLS. These models are used for displaying Constitutively Activating Mutations.

This tool is to investigate potential interrelationships between wild type amino acids or mutations. Interacting residues are likely to cause similar phenotypes if they're subjected to similar mutagenesis experiments. The projection feature gives the opportunity to investigate such mechanisms and also hot-spots of functionalities.

In case of a functional parameter search, similar to the result table there are highlighting controls available. They allow an individual classification scheme, which can be visualised on the receptor. Additionally there is an option for projecting all mutations on the receptor.

Three-dimensional morphing at receptor models: from the basal to the activated receptor conformation

The 3d morph applet shows a morph between an inactivated conformation (based on the X-ray structure of bovine rhodopsin) and an activated conformation (based on the X-ray structure of opsin). Presented are the wildtype side-chains of the mutated positions. To zoom to the presented mutated position, tick the appropriate box. The yellow box in the left upper corner shows whether the conformation is inactivated or activated. The interpolation and energy minimization for the morph were done by the Morph Server of Gerstein & Krebs at Yale Univ.

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